Figure 3JNK - induced transcription factors are critically required for EMT . ( A ) Venn diagram showing overlap of genes upregulated upon JNKi treatment during EMT and downregulated during normal EMT at day 7 that contains 5 transcription factors . Right panel shows RNA levels ( normalized tag counts ) for these factors . ( B ) Same overlap as in ( A ) for genes downregulated upon JNKi treatment during EMT and upregulated during normal EMT . Right panel displays RNA levels as in ( A ) for 10 transcription factors identified in this overlap . ( C ) Representative bright field and immunofluorescence images for localization and expression levels of EMT marker proteins after four days ( d4 ) of siRNA - mediated depletion of factors compared to non - targeting control ( NTC ) . Staining was performed with antibodies against epithelial markers E - cadherin and ZO1 , mesenchymal markers Fibronectin 1 , with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques . Scale bar , 100 μm , 40X magnification . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3JNK-induced transcription factors are critically required for EMT. (A) Venn diagram showing overlap of genes upregulated upon JNKi treatment during EMT and downregulated during normal EMT at day 7 that contains 5 transcription factors. Right panel shows RNA levels (normalized tag counts) for these factors.(B) Same overlap as in (A) for genes downregulated upon JNKi treatment during EMT and upregulated during normal EMT. Right panel displays RNA levels as in (A) for 10 transcription factors identified in this overlap.(C) Representative bright field and immunofluorescence images for localization and expression levels of EMT marker proteins after four days (d4) of siRNA-mediated depletion of factors compared to non-targeting control (NTC). Staining was performed with antibodies against epithelial markers E-cadherin and ZO1, mesenchymal markers Fibronectin 1, with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques. Scale bar, 100 μm, 40X magnification. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


Figure 4Depletion of Prkrir , Pcbp4 and Tsc22d1 rescues TGF - β - induced reduction in the rate of cell division . ( A ) Lineage trees tracked by live imaging under control , Non treated control ( NTC ) and siRNA - mediated depletion of factors ( X = cell death / cells could not be tracked till the end ) . ( B ) Average number of rounds of division per clone in NTC compared with control and siRNA - mediated depletion of factors ( ANOVA , Tukeys post - test , * = p < 0 . 05 , * * = p < 0 . 01 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Depletion of Prkrir, Pcbp4 and Tsc22d1 rescues TGF-β-induced reduction in the rate of cell division. (A) Lineage trees tracked by live imaging under control, Non treated control (NTC) and siRNA-mediated depletion of factors (X= cell death/cells could not be tracked till the end).(B) Average number of rounds of division per clone in NTC compared with control and siRNA-mediated depletion of factors (ANOVA, Tukeys post-test, *=p<0.05,**=p<0.01).


Figure 5PRKRIR , PCBP4 and TSC22D1 are crucial for maintenance of the mesenchymal fate . ( A ) Scratch assay in MDA - MB - 231 cells treated for four days with DMSO or JNKi . Scale bar , 200 μm ; 20 X magnification . Mean and SEM is plotted from three independent biological replicates . ( B ) mRNA levels for key EMT marker genes in MDA - MB - 231 treated with DMSO or JNKi for 4 days were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates ( C ) mRNA levels for transcription factors in isogenic epithelial ( MDA - MB - 361 ) and mesenchymal ( MDA - MB - 231 ) breast cancer cells were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates . ( D ) Scratch assay in MDA - MB - 231 cells transfected with NTC or siRNA against PRKRIR , NFIL3 , PCBP4 and TSC22D1 for 4 days as in A . Mean and SEM is plotted from three independent biological replicates as bar plot on right . ( E ) Invasion assay in MDA - MB - 231 cells treated as in ( D ) . Mean and SEM is plotted from three independent biological replicates . ( F ) mRNA levels of key EMT marker genes in MDA - MB - 231 and MDA - MB - 231 transfected with either empty vector or vector overexpressing PCBP4 or TSC22D1 for 48 hours and then treated with the JNK inhibitor ( SP600125 ) for 24 hours were measured by qRT - PCRs relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5PRKRIR, PCBP4 and TSC22D1 are crucial for maintenance of the mesenchymal fate. (A) Scratch assay in MDA-MB-231 cells treated for four days with DMSO or JNKi. Scale bar, 200 μm; 20 X magnification. Mean and SEM is plotted from three independent biological replicates.(B) mRNA levels for key EMT marker genes in MDA-MB-231 treated with DMSO or JNKi for 4 days were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates(C) mRNA levels for transcription factors in isogenic epithelial (MDA-MB-361) and mesenchymal (MDA-MB-231) breast cancer cells were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates.(D) Scratch assay in MDA-MB-231 cells transfected with NTC or siRNA against PRKRIR, NFIL3, PCBP4 and TSC22D1 for 4 days as in A. Mean and SEM is plotted from three independent biological replicates as bar plot on right .(E) Invasion assay in MDA-MB-231 cells treated as in (D). Mean and SEM is plotted from three independent biological replicates.(F) mRNA levels of key EMT marker genes in MDA-MB-231 and MDA-MB-231 transfected with either empty vector or vector overexpressing PCBP4 or TSC22D1 for 48 hours and then treated with the JNK inhibitor (SP600125) for 24 hours were measured by qRT-PCRs relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


Figure 6 ( C ) mRNA levels for representative genes highlighted in ( A ) for cells treated like in ( Fig 5D ) were measured by qRT - PCR relative to Ctcf and plotted on the y - axis . Mean and SEM is plotted from three independent biological replicates . F ) Representative immunofluorescence images showing localization of Tsc22d1 in NMuMG cells following transient expression of Flag - HA tagged Tsc22d1 and detection using an anti - Flag antibody , Bar plot shows quantification of Tsc22d1 localization with respect to DAPI density within the nucleus . Y - axis shows the percentage of total fluorescence intensity for Tsc22d1 in DAPI dense and light regions . SEM is derived from independent biological replicates . ( G ) ChIP assay using anti - Flag antibody following expression of Flag - HA tagged TSC22D1 in MDA - MB - 231 cells . Quantitative PCRs were performed for indicated gene promoters and enrichments are plotted on the y - axis as ratio of precipitated DNA ( bound ) to total input DNA and then further divided by the same obtained in the empty vector transfected cells . SEM is derived from independent biological replicates . ( H ) mRNA levels for PRKRIR , PCBP4 , TSC22D1 in non - invasive ( DCIS ) ( n = 20 ) and invasive tumor ( n = 20 ) samples were measured by qRT PCRs relative to TBP and plotted on the y - axis . ZEB1 was used as an established positive control for invasive tumors . Plotted Mean ± SEM of three biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(C) mRNA levels for representative genes highlighted in (A) for cells treated like in (Fig 5D) were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates.F) Representative immunofluorescence images showing localization of Tsc22d1 in NMuMG cells following transient expression of Flag-HA tagged Tsc22d1 and detection using an anti-Flag antibody, Bar plot shows quantification of Tsc22d1 localization with respect to DAPI density within the nucleus. Y-axis shows the percentage of total fluorescence intensity for Tsc22d1 in DAPI dense and light regions. SEM is derived from independent biological replicates.(G) ChIP assay using anti-Flag antibody following expression of Flag-HA tagged TSC22D1 in MDA-MB-231 cells. Quantitative PCRs were performed for indicated gene promoters and enrichments are plotted on the y-axis as ratio of precipitated DNA (bound) to total input DNA and then further divided by the same obtained in the empty vector transfected cells. SEM is derived from independent biological replicates.(H) mRNA levels for PRKRIR, PCBP4, TSC22D1 in non-invasive (DCIS) (n=20) and invasive tumor (n=20) samples were measured by qRT PCRs relative to TBP and plotted on the y-axis. ZEB1 was used as an established positive control for invasive tumors. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


Figure 7Newly identified transcription factors are similarly upregulated during neurogenesis and function in neuronalmigration . ( A ) RT - qPCR analysis for key EMT markers in embryonic stem cells ( ES ) , neuronal progenitors ( NP ) and terminally differentiated neurons ( TN ) . mRNA levels were measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates . ( B ) Western blot analyzing total JNK and phospho - JNK ( p - JNK ) during neuronal differentiation of stem cells . β - actin serve as a loading control . ( C ) RNA levels of Pcbp4 , Prkrir and Tsc22d1 during neuronal differentiation as shown in A , measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates . ( D ) RNA levels of Pcbp4 , Prkrir , Tsc22d1 , represented by average counts derived from previously published microarray data of independent biological replicates from neurons treated with DMSO or JNK inhibitor SP600125 ( JNKi ) for 6 hours . ( E ) RNA levels of Tsc22d1 and three Tsc22d1 target genes during various stages of neuronal differentiation shown in ( A ) , measured by RT - qPCR relative to Ctcf and plotted on the y - axis . Error bars represent SEM from independent biological replicates . ( F ) RNA levels of Tsc22d1 and the same three target genes is shown as average normalized tag counts derived from RNA - seq data from Ventricular Zone ( VZ ) , Sub - Ventricular Zone ( SVZ ) and Cortical plate ( CP ) of E14 . 5 mouse Cortex . ( G ) RNA levels of genes shown in Figure ( E ) , represented by average normalized tag counts derived from RNA - seq data of TGF - β - induced EMT in NMuMG cells and plotted on the y - axis . Error bars represent SEM from three biological replicates . ( I ) A representative image from immunofluorescence analysis performed with anti - GFP and anti - Tbr1 antibody that shows retention of GFP positive cells below the Tbr1 layer in Tsc22d1 knockdown brain as compared to the control brain . The bar plot on the right side shows quantification of migrated GFP positive cells in control and Tsc22d1 depleted mouse brain with respect to the Tbr1 staining . y axis represent percentage of cells above or below the Tbr1 stain region . Error bars represent SEM from 3 independent biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , Student ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Newly identified transcription factors are similarly upregulated during neurogenesis and function in neuronalmigration. (A) RT-qPCR analysis for key EMT markers in embryonic stem cells (ES), neuronal progenitors (NP) and terminally differentiated neurons (TN). mRNA levels were measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.(B) Western blot analyzing total JNK and phospho-JNK (p-JNK) during neuronal differentiation of stem cells. β-actin serve as a loading control.(C) RNA levels of Pcbp4, Prkrir and Tsc22d1 during neuronal differentiation as shown in A, measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.(D) RNA levels of Pcbp4, Prkrir, Tsc22d1, represented by average counts derived from previously published microarray data of independent biological replicates from neurons treated with DMSO or JNK inhibitor SP600125 (JNKi) for 6 hours.(E) RNA levels of Tsc22d1 and three Tsc22d1 target genes during various stages of neuronal differentiation shown in (A), measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates.(F) RNA levels of Tsc22d1 and the same three target genes is shown as average normalized tag counts derived from RNA-seq data from Ventricular Zone (VZ), Sub-Ventricular Zone (SVZ) and Cortical plate (CP) of E14.5 mouse Cortex.(G) RNA levels of genes shown in Figure (E), represented by average normalized tag counts derived from RNA-seq data of TGF-β-induced EMT in NMuMG cells and plotted on the y-axis. Error bars represent SEM from three biological replicates.(I) A representative image from immunofluorescence analysis performed with anti-GFP and anti-Tbr1 antibody that shows retention of GFP positive cells below the Tbr1 layer in Tsc22d1 knockdown brain as compared to the control brain. The bar plot on the right side shows quantification of migrated GFP positive cells in control and Tsc22d1 depleted mouse brain with respect to the Tbr1 staining. y axis represent percentage of cells above or below the Tbr1 stain region. Error bars represent SEM from 3 independent biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student's t-test.


Figure 2 ( C ) To assign identities to these germ cell subclusters , the violin plots were used to visualize the distribution of normalized typical marker genes expression levels . ( E ) The expression of GSC maintenance genes ( aret , Mei - P26 , egg , Myc and Hrb98DE ) and differentiation genes ( bam , bgcn , out , twin and Set1 ) along the primary branch ( dotted circle line ) in pseudotime .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(C) To assign identities to these germ cell subclusters, the violin plots were used to visualize the distribution of normalized typical marker genes expression levels.(E) The expression of GSC maintenance genes (aret, Mei-P26, egg, Myc and Hrb98DE) and differentiation genes (bam, bgcn, out, twin and Set1) along the primary branch (dotted circle line) in pseudotime.


Figure 3 ( C ) The typical phenotypes of germaria in selected RNAi lines stained with anti - α - Spectrin ( green ) and anti - pMad ( red ) . The scale bar is 10 μm . ( E ) The heat map of SCENIC analysis of the GSC , undifferentiated germ cell - 1 , undifferentiated germ cell - 2 , early nurse cell , oocyte and later nurse cell subclusters .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(C) The typical phenotypes of germaria in selected RNAi lines stained with anti-α-Spectrin (green) and anti-pMad (red). The scale bar is 10 μm.(E) The heat map of SCENIC analysis of the GSC, undifferentiated germ cell-1, undifferentiated germ cell-2, early nurse cell, oocyte and later nurse cell subclusters.


Figure 4 ( B ) The expression patterns of candidate marker genes were validated by using in situ hybridization . The scale bar is 10 μm . ( C - C ' ) Immunofluorescence staining with anti - TfIIA - S on wild type and nos - Gal4 > UAS - TfIIA - S - RNAi ( negative control ) . The scale bar is 10 μm . ( E - E ' ) Immunofluorescence staining with anti - eggpl on wild type and nos - Gal4 > UAS - eggpl - RNAi ( negative control ) ovary . The scale bar is 10 μm . ( F ) The overexpression of GFP ( green ) and eggpl in Sf9 cell line in vitro . The anti - eggpl ( red ) was used to detect the eggpl protein , and Hoechst ( blue ) was used to label the nucleus . The scale bar is 10 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) The expression patterns of candidate marker genes were validated by using in situ hybridization. The scale bar is 10 μm.(C-C') Immunofluorescence staining with anti-TfIIA-S on wild type and nos-Gal4>UAS-TfIIA-S-RNAi (negative control). The scale bar is 10 μm.(E-E') Immunofluorescence staining with anti-eggpl on wild type and nos-Gal4>UAS-eggpl-RNAi (negative control) ovary. The scale bar is 10 μm.(F) The overexpression of GFP (green) and eggpl in Sf9 cell line in vitro. The anti-eggpl (red) was used to detect the eggpl protein, and Hoechst (blue) was used to label the nucleus. The scale bar is 10 μm.


Figure 5 ( A - A ' ) The mRNA in situ hybridization of eggpl ( green ) on wild type and nos - Gal4 > UAS - eggpl - RNAi ( negative control ) line . The scale bar is 10 μm . ( B - C ) Immunofluorescence staining with anti - α - Spectrin ( red ) and anti - Vasa ( red ) on eggpl - in situ ( green ) labeled tissues respectively . The scale bar is 10 μm . The dashed line indicates the eggpl - in situ ( green ) signals . ( F and H ) The mRNA in situ hybridization of eggpl ( green ) on nos - Gal4 > UAS - eggpl - RNAi ( negative control ) ( F ) and nos - Gal4 > UAS - eggpl - GFP lines ( H ) . The scale bar is 10 μm . ( G ) The anti - GFP , anti - α - Spectrin ( red ) and anti - Bam ( blue ) were used to stain on eggpl : : GFP knock - in line . The scale bar is 10 μm . ( I ) Double staining with eggpl in situ and anti - GFP on eggpl knock - in lines . The scale bar is 10 μm . The dashed line indicates the eggpl - in situ ( green ) signals . ( K ) The phenotypes of wild type , nos - Gal4 > UAS - eggpl - RNAi line and nos - Gal4 > UAS - eggpl : : GFP line staining with anti - α - Spectrin ( green ) , anti - pMad ( red ) . The scale bar is 10 μm . ( L ) The average number of GSC and CB in nos - Gal4 line , nos - Gal4 > UAS - eggpl - RNAi line and nos - Gal4 > UAS - eggpl : : GFP line on 2 day , 7 day , 14 day and 21 day . Error bars show mean ± SEM , one - way ANOVA , ns indicates no significant difference , * P < 0 . 05 , * * * P < 0 . 001 , n ≥ 72 , biological replicates . ( Q ) The statistical analysis of the average number of GSCs and CBs and Cysts in eggpl [ 1 ] line . Error bars show mean ± SEM , one - way ANOVA , ns indicates no significant difference , * * * P < 0 . 001 , n ≥ 104 , biological replicates . ( R ) The average number of apoptotic germ cells in wild type , nos - Gal4 > UAS - eggpl - RNAi line and eggpl [ 1 ] line . Error bars show mean ± SEM , one - way ANOVA , ns indicates no significant difference ( biological replicates , n = 3 ) , and N represents the total number of counted ovarioles .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A-A') The mRNA in situ hybridization of eggpl (green) on wild type and nos-Gal4>UAS-eggpl-RNAi (negative control) line. The scale bar is 10 μm. (B-C) Immunofluorescence staining with anti-α-Spectrin (red) and anti-Vasa (red) on eggpl-in situ (green) labeled tissues respectively. The scale bar is 10 μm. The dashed line indicates the eggpl-in situ (green) signals.(F and H) The mRNA in situ hybridization of eggpl (green) on nos-Gal4>UAS-eggpl-RNAi (negative control) (F) and nos-Gal4>UAS-eggpl-GFP lines (H). The scale bar is 10 μm. (G) The anti-GFP, anti-α-Spectrin (red) and anti-Bam (blue) were used to stain on eggpl::GFP knock-in line. The scale bar is 10 μm. (I) Double staining with eggpl in situ and anti-GFP on eggpl knock-in lines. The scale bar is 10 μm. The dashed line indicates the eggpl-in situ (green) signals.(K) The phenotypes of wild type, nos-Gal4>UAS-eggpl-RNAi line and nos-Gal4>UAS-eggpl::GFP line staining with anti-α-Spectrin (green), anti-pMad (red). The scale bar is 10 μm.(L) The average number of GSC and CB in nos-Gal4 line, nos-Gal4>UAS-eggpl-RNAi line and nos-Gal4>UAS-eggpl::GFP line on 2 day, 7 day, 14 day and 21 day. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference, *P<0.05, ***P<0.001, n ≥ 72, biological replicates.(Q) The statistical analysis of the average number of GSCs and CBs and Cysts in eggpl[1] line. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference, ***P<0.001, n ≥ 104, biological replicates.(R) The average number of apoptotic germ cells in wild type, nos-Gal4>UAS-eggpl-RNAi line and eggpl[1] line. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference (biological replicates, n=3), and N represents the total number of counted ovarioles.


Figure 6 ( A ) Immunofluorescence staining with anti - α - Spectrin ( red ) , anti - Vasa ( blue ) and anti - GFP ( green ) on adult ovary . The scale bar is 10 μm . ( B ) Immunofluorescence staining on PGCs of larvae ovary . The scale bar is 20 μm . ( C ) Immunofluorescence staining on GSCs and early spermatogonia in adult testis . The scale bar is 20 μm . ( D ) Immunofluorescence staining on PGCs of larvae testis tissue . The scale bar is 20 μm . ( E ) Immunofluorescence staining with anti - α - Spectrin ( red ) and anti - Vasa ( green ) on testis from wild type or eggpl [ 1 ] flies . The scale bar is 10 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Immunofluorescence staining with anti-α-Spectrin (red), anti-Vasa (blue) and anti-GFP (green) on adult ovary. The scale bar is 10 μm. (B) Immunofluorescence staining on PGCs of larvae ovary. The scale bar is 20 μm. (C) Immunofluorescence staining on GSCs and early spermatogonia in adult testis. The scale bar is 20 μm.(D) Immunofluorescence staining on PGCs of larvae testis tissue. The scale bar is 20 μm. (E) Immunofluorescence staining with anti-α-Spectrin (red) and anti-Vasa (green) on testis from wild type or eggpl[1] flies. The scale bar is 10 μm.


Figure 7 ( A ) Immunofluorescence staining with anti - BrdU ( red ) . The scale bar is 10 μm . ( B ) The oviposition of wild type , Nos - Gal4 > UAS - eggpl - RNAi and eggpl [ 1 ] line over 21 days . Error bars show mean ± SEM , 3 biological replicates ( n = 3 ) were performed , and there were 10 female and 5 male adult flies in each biological replicate . ( F ) Immunofluorescence staining with anti - GFP on germaria from eggpl : : GFP knock - in flies with or without feeding fresh yeast paste on day 2 - , 7 - , 14 - and 21 . The dotted line indicates the examined area . The scale bar is 10 μm . ( G ) Quantification of the GFP intensity mean in the eggpl : : GFP knock - in line . The central band indicates the median , the box length indicates the interquartile range , and the upper and whisker indicate the maximum and minimum value respectively . The 10 biological replicates ( n = 10 ) were performed , one - way ANOVA , * * * P < 0 . 001 . The dashed line indicates the examined area . ( H - I ) Images of ovaries or mature eggs from flies of the indicated genotypes , feeding conditions , and days post - eclosion showing comparisons of the overall ovary sizes ( F ) and the numbers of retained matured eggs ( G ) . The scar bar is 500 μm . ( J - K ) Quantitative analysis of the ovary size ( H ) and the number of retained mature eggs . ( I ) under different food conditions . Error bars show mean ± SEM , one - way ANOVA , ns indicates no significant difference , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . The 3 biological replicates ( n = 3 ) were performed .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Immunofluorescence staining with anti-BrdU (red). The scale bar is 10 μm.(B) The oviposition of wild type, Nos-Gal4>UAS-eggpl-RNAi and eggpl[1] line over 21 days. Error bars show mean ± SEM, 3 biological replicates (n=3) were performed, and there were 10 female and 5 male adult flies in each biological replicate.(F) Immunofluorescence staining with anti-GFP on germaria from eggpl::GFP knock-in flies with or without feeding fresh yeast paste on day 2-, 7-, 14- and 21. The dotted line indicates the examined area. The scale bar is 10 μm. (G) Quantification of the GFP intensity mean in the eggpl::GFP knock-in line. The central band indicates the median, the box length indicates the interquartile range, and the upper and whisker indicate the maximum and minimum value respectively. The 10 biological replicates (n=10) were performed, one-way ANOVA, ***P<0.001. The dashed line indicates the examined area.(H-I) Images of ovaries or mature eggs from flies of the indicated genotypes, feeding conditions, and days post-eclosion showing comparisons of the overall ovary sizes (F) and the numbers of retained matured eggs (G). The scar bar is 500 μm. (J-K) Quantitative analysis of the ovary size (H) and the number of retained mature eggs. (I) under different food conditions. Error bars show mean ± SEM, one-way ANOVA, ns indicates no significant difference, *P<0.05, **P<0.01, ***P<0.001. The 3 biological replicates (n=3) were performed.


Figure 8 ( B ) Quantification of the GFP signal in germaria from eggpl : : GFP knock - in flies of the indicated genotypes under different conditions . The central band indicates the median , the box length indicates the interquartile range , and the upper and whisker indicate the maximum and minimum value respectively , one - way ANOVA , * * P < 0 . 01 , * * * P < 0 . 001 , n = 10 , biological replicates . ( C ) Immunofluorescence staining with anti - GFP on germaria with or without feeding fresh yeast paste on 7th day . The dashed line indicates the examined area . The scale bar is 10 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(B) Quantification of the GFP signal in germaria from eggpl::GFP knock-in flies of the indicated genotypes under different conditions. The central band indicates the median, the box length indicates the interquartile range, and the upper and whisker indicate the maximum and minimum value respectively, one-way ANOVA, **P<0.01, ***P<0.001, n = 10, biological replicates.(C) Immunofluorescence staining with anti-GFP on germaria with or without feeding fresh yeast paste on 7th day. The dashed line indicates the examined area. The scale bar is 10 μm.


Figure 1A . Immunoblotting of c - MYC and ATF4 in melanoma cells treated with NT - siRNA ( - ) or siASNS ( # 1 or # 2 ) for 72 hr . Reverse transcription with quantitative PCR ( RT - qPCR ) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS ( # 1 or # 2 ) ( B ) C . Reverse transcription with quantitative PCR ( RT - qPCR ) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS , siATF4 , or both ( C ) . D . Immunoblotting of ATF4 and ASNS in melanoma cells 72 hr after treatment with siASNS , siATF4 , or both . E . Proliferation of melanoma cells over indicated times after treatment with siASNS , siATF4 , or both . F . RT - qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS ( # 1 or # 2 ) . Immunoblotting of c - MYC in melanoma cells treated for 72 hr with siASNS , L - Asn , or both ( G ) H . Immunoblotting of c - MYC in A375 cells treated as indicated for 72 hr ( H ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O

Figure 1A. Immunoblotting of c-MYC and ATF4 in melanoma cells treated with NT-siRNA (-) or siASNS (#1 or #2) for 72 hr.Reverse transcription with quantitative PCR (RT-qPCR) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2) (B)C. Reverse transcription with quantitative PCR (RT-qPCR) of the indicated transcripts in melanoma cells 36 hr after treatment with siASNS, siATF4, or both (C).D. Immunoblotting of ATF4 and ASNS in melanoma cells 72 hr after treatment with siASNS, siATF4, or both.E. Proliferation of melanoma cells over indicated times after treatment with siASNS, siATF4, or both.F. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS (#1 or #2).Immunoblotting of c-MYC in melanoma cells treated for 72 hr with siASNS, L-Asn, or both (G)H. Immunoblotting of c-MYC in A375 cells treated as indicated for 72 hr (H).


Figure 2A . Immunoblotting of c - MYC in A375 cells treated with NT - siRNA or siASNS for 24 hr followed by cycloheximide treatment for indicated timepoints ( left ) . Plot showing relative protein levels of c - MYC ( y - axis ) as a function of time lapsed ( x - axis ) in the left panel ( right ) . Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with NT - siRNA ( - ) or siASNS ( # 1 or # 2 ) ( B and C ) Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS , PLX - 4032 , or both ( D ) . E . Immunoblotting of indicated proteins in A375 cells cultured in L - Asn and treated with L - A ' ase , PLX - 4032 , or both for 72 hr . F . A375 cells were treated with siASNS , SCH772984 , or both for 72 hr followed by immunoblotting of phosphorylated and total GSK3 - β . G . A375 cells were treated with siASNS , HA - GSK3 - β ( S9A ) , or both for 72 hr followed by immunoblotting of indicated proteins . H . RT - qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both . I . A375 cells were treated as indicated for 72 hr followed by immunoblotting of c - MYC . J . Immunoblotting of c - MYC and HA proteins in A375 cells treated as indicated for 72 hr .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O

Figure 2A. Immunoblotting of c-MYC in A375 cells treated with NT-siRNA or siASNS for 24 hr followed by cycloheximide treatment for indicated timepoints (left). Plot showing relative protein levels of c-MYC (y-axis) as a function of time lapsed (x-axis) in the left panel (right).Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with NT-siRNA (-) or siASNS (#1 or #2) (B and C)Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, PLX-4032, or both (D).E. Immunoblotting of indicated proteins in A375 cells cultured in L-Asn and treated with L-A'ase, PLX-4032, or both for 72 hr.F. A375 cells were treated with siASNS, SCH772984, or both for 72 hr followed by immunoblotting of phosphorylated and total GSK3-β.G. A375 cells were treated with siASNS, HA-GSK3-β (S9A), or both for 72 hr followed by immunoblotting of indicated proteins.H. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS, HA-GSK3-β (S9A), or both.I. A375 cells were treated as indicated for 72 hr followed by immunoblotting of c-MYC.J. Immunoblotting of c-MYC and HA proteins in A375 cells treated as indicated for 72 hr.


Figure 3A . Immunoblotting of indicated proteins in melanoma cells treated with siASNS , sicMYC ( # 1 or # 2 ) , or a combination of siASNS and sicMYC ( # 1 or # 2 ) for 72 hr . B . RT - qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS , sicMYC , or both . C . Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS , sicMYC , PLX - 4032 , or a combination of siASNS and sicMYC , or siASNS and PLX - 4032 . D . Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both . E . RT - qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS , HA - GSK3 - β ( S9A ) , or both . F . A375 cells were treated as indicated for 72 hr followed by immunoblotting of indicated proteins . Proliferation of melanoma cells over indicated times after treatment with siASNS , sicMYC , or both ( G ) , Proliferation of melanoma cells over indicated times after treatment with siASNS , HA - GSK3 - β ( S9A ) , or both ( H ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Immunoblotting of indicated proteins in melanoma cells treated with siASNS, sicMYC (#1 or #2), or a combination of siASNS and sicMYC (#1 or #2) for 72 hr.B. RT-qPCR analysis of indicated transcripts in melanoma cells 36 hr after treatment with siASNS, sicMYC, or both.C. Immunoblotting of indicated proteins in melanoma cells 72 hr after treatment with siASNS, sicMYC, PLX-4032, or a combination of siASNS and sicMYC, or siASNS and PLX-4032.D. Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS, HA-GSK3-β (S9A), or both.E. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, HA-GSK3-β (S9A), or both.F. A375 cells were treated as indicated for 72 hr followed by immunoblotting of indicated proteins.Proliferation of melanoma cells over indicated times after treatment with siASNS, sicMYC, or both (G),Proliferation of melanoma cells over indicated times after treatment with siASNS, HA-GSK3-β (S9A), or both (H).


Figure 4A . Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS , siSLC1A5 , siSLC7A5 , or a combination of siASNS and siSLC1A5 , or siASNS and siSLC7A5 . B . RT - qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS , siSLC7A5 , or both . C . Proliferation of melanoma cells over indicated times after treatment with siASNS , siSLC7A5 , or both . D . GC - MS - based estimation of intracellular amino acid levels in A375 cells treated with siASNS , siATF4 , or both ( top left ) ; siASNS , sicMYC , or both ( top right ) ; siASNS , siSLC1A5 , or both ( bottom left ) ; or siASNS , siSLC7A5 , or both ( bottom right ) . The fold change is relative to NT - siRNA - treated A375 cells . Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment ( E ) , Immunoblotting of indicated proteins in A375 cells 72 hr after , treatment with siASNS , sicMYC , or bothImmunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS , siSLC7A5 , or both ( G ) Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS , BCH , or both ( H ) Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS , HA - GSK3 - β ( S9A ) , or both ( I ) Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS or indicated combinations ( J ) Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS , siSLC3A2 , or both ( K ) . L . RT - qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS , siSLC3A2 , or both . M . Proliferation of melanoma cells over indicated times after treatment with siASNS , siSLC3A2 , or both .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 4A. Immunoblotting of indicated proteins in A375 cells 72 hr after treatment with siASNS, siSLC1A5, siSLC7A5, or a combination of siASNS and siSLC1A5, or siASNS and siSLC7A5.B. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, siSLC7A5, or both.C. Proliferation of melanoma cells over indicated times after treatment with siASNS, siSLC7A5, or both.D. GC-MS-based estimation of intracellular amino acid levels in A375 cells treated with siASNS, siATF4, or both (top left); siASNS, sicMYC, or both (top right); siASNS, siSLC1A5, or both (bottom left); or siASNS, siSLC7A5, or both (bottom right). The fold change is relative to NT-siRNA-treated A375 cells.Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment (E),Immunoblotting of indicated proteins in A375 cells 72 hr after , treatment with siASNS, sicMYC, or bothImmunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS, siSLC7A5, or both (G)Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, BCH, or both (H)Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, HA-GSK3-β (S9A), or both (I)Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with , siASNS or indicated combinations (J)Immunoblotting of indicated proteins in A375 cells 72 hr after indicated treatment with siASNS, siSLC3A2, or both (K).L. RT-qPCR analysis of indicated transcripts in melanoma cells after 36 hr treatment with siASNS, siSLC3A2, or both.M. Proliferation of melanoma cells over indicated times after treatment with siASNS, siSLC3A2, or both.


Figure 1A . Confocal microscopy image of PD myotubes ( cl . 3LE8 ) infected for 72 h with adenovirus containing TFEB ( PD + Ad - TFEB 72 h ) shows a dramatic reduction in the number of large LAMP1 - positive lysosomes ( red ) compared to that in untreated ( PD ) or adenovirus ( PD + Ad - null ) - treated PD myotubes . WT myotubes are shown on the left panel . Nuclei are stained with Hoechst ( blue ) . TFEB was detected with anti - Flag antibody ( green ) . B . Distribution of lysosomal size differs significantly in Ad - null and Ad - TFEB PD myotubes ( p = 6 . 32 × 10 − 8 ; Kolmogorov - Smirnov test ) . Lysosomal size is expressed as number of pixels representing lysosomal area ( LAMP1 - positive structures ) . The median lysosomal size of Ad - TFEB infected myotubes ( m = 367 . 13 pixels , n = 703 , range 208 - 2659 ) was significantly lower than that of Ad - null infected myotubes ( m = 491 . 16 pixels , n = 1395 , range 200 - 2857 ; p = 6 . 5 × 10 − 12 ; Wilcoxon rank sum test ) . C . Confocal microscopy image of PD myotubes infected for 24 h with Ad - TFEB shows relocation of lysosomes to the plasma membrane ( top ) . Images showing LAMP1 staining ( red ) on plasma membrane in a PD myotube infected with Ad - TFEB ( bottom ; arrows ) but not in a non - infected cell ( middle ) . Non - permeabilized cells were incubated with anti - LAMP1 antibody at 4 ° C for 40 min , followed by fixation and staining with secondary antibody . D . Confocal microscopy images of live non - infected PD myotubes ( left ) or PD myotubes infected for 72 h with Ad - TFEB ( right ) show a dramatic reduction in the amount of accumulated glycogen in TFEB - treated cells . The cells were incubated with the fluorescent glucose ( 2 - NBDG ; green ) , extensively washed , and analysed by confocal microscopy . Bar : 10 µm for all panels .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A. Confocal microscopy image of PD myotubes (cl. 3LE8) infected for 72 h with adenovirus containing TFEB (PD + Ad-TFEB 72 h) shows a dramatic reduction in the number of large LAMP1-positive lysosomes (red) compared to that in untreated (PD) or adenovirus (PD + Ad-null)-treated PD myotubes. WT myotubes are shown on the left panel. Nuclei are stained with Hoechst (blue). TFEB was detected with anti-Flag antibody (green).B. Distribution of lysosomal size differs significantly in Ad-null and Ad-TFEB PD myotubes (p = 6.32 × 10−8; Kolmogorov-Smirnov test). Lysosomal size is expressed as number of pixels representing lysosomal area (LAMP1-positive structures). The median lysosomal size of Ad-TFEB infected myotubes (m = 367.13 pixels, n = 703, range 208-2659) was significantly lower than that of Ad-null infected myotubes (m = 491.16 pixels, n = 1395, range 200-2857; p = 6.5 × 10−12; Wilcoxon rank sum test).C. Confocal microscopy image of PD myotubes infected for 24 h with Ad-TFEB shows relocation of lysosomes to the plasma membrane (top). Images showing LAMP1 staining (red) on plasma membrane in a PD myotube infected with Ad-TFEB (bottom; arrows) but not in a non-infected cell (middle). Non-permeabilized cells were incubated with anti-LAMP1 antibody at 4°C for 40 min, followed by fixation and staining with secondary antibody.D. Confocal microscopy images of live non-infected PD myotubes (left) or PD myotubes infected for 72 h with Ad-TFEB (right) show a dramatic reduction in the amount of accumulated glycogen in TFEB-treated cells. The cells were incubated with the fluorescent glucose (2-NBDG; green), extensively washed, and analysed by confocal microscopy. Bar: 10 µm for all panels.


Figure 2Confocal microscopy of live unstained fibres from a GFP - LC3 : WT mouse ( WT ) and from untreated ( GAA − / − ) or ERT - treated ( GAA − / − ; + ERT ) GFP - LC3 : GAA − / − mice . Images show typical centrally located autophagic build - up with multiple clusters of LC3 - positive autophagosomes . Structures like these are never seen in WT muscle . Autophagic build - up is present virtually in all fibres derived from EDL or gastrocnemiusmuscles ( not shown ) , but only in 60 - 70 % of FDBfibres . Labelled recombinant human GAA ( red ) was administered i . v . into 3 - 4 month - old GFP - LC3 : GAA − / − mice ( n = 5 ) at a dose of 100 mg / kg twice with a 24 h interval . Mice were sacrificed the next day , and live fibres ( shown for EDLmuscle ) were analysed by confocal microscopy . The labelled recombinant GAA was detected almost exclusively in the autophagic vesicles . At least 500 fibres were analysed for each condition . Bar : 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Confocal microscopy of live unstained fibres from a GFP-LC3:WT mouse (WT) and from untreated (GAA−/−) or ERT-treated (GAA−/−; +ERT) GFP-LC3:GAA−/− mice. Images show typical centrally located autophagic build-up with multiple clusters of LC3-positive autophagosomes. Structures like these are never seen in WT muscle. Autophagic build-up is present virtually in all fibres derived from EDL or gastrocnemiusmuscles (not shown), but only in 60-70% of FDBfibres. Labelled recombinant human GAA (red) was administered i.v. into 3-4 month-old GFP-LC3:GAA−/− mice (n = 5) at a dose of 100 mg/kg twice with a 24 h interval. Mice were sacrificed the next day, and live fibres (shown for EDLmuscle) were analysed by confocal microscopy. The labelled recombinant GAA was detected almost exclusively in the autophagic vesicles. At least 500 fibres were analysed for each condition. Bar: 10 µm.


Figure 3A . Immunostaining of non - infected cells ( day 13 in differentiation medium ) and cells infected with a mutant form of TFEB ( TFEBmt ) with LAMP1 ( red ) and Flag ( green ) . Ad - TFEBmt was added to the myotubes for 72 h on day 10 in differentiation medium . Ad - TFEBmt - infected cells show massive accumulation of TFEB in the nuclei and significant reduction in lysosomal size , similar to that seen with TFEB . B . Western blot of cell lysates confirms the presence of TFEB in the nuclear fraction ; the different intensities of the two bands corresponding to LAMP1 protein in untreated and TFEBmt - treated samples may reflect the differences in the glycosylation pattern . C . Western blot of protein lysates from untreated , Ad - null , TFEB , and TFEBmt - treated myotubes with anti - caspase - 3 antibody . No activated ( cleaved ) products are detected in any condition . α - Tubulin was used as a loading control . D . TUNEL assay shows the presence of some apoptotic cells in Ad - TFEB - infected cultures , but not in control cultures infected with adenovirus alone ( n = 3 ) . Bar : 10 µm for all panels .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A. Immunostaining of non-infected cells (day 13 in differentiation medium) and cells infected with a mutant form of TFEB (TFEBmt) with LAMP1 (red) and Flag (green). Ad-TFEBmt was added to the myotubes for 72 h on day 10 in differentiation medium. Ad-TFEBmt-infected cells show massive accumulation of TFEB in the nuclei and significant reduction in lysosomal size, similar to that seen with TFEB.B. Western blot of cell lysates confirms the presence of TFEB in the nuclear fraction; the different intensities of the two bands corresponding to LAMP1 protein in untreated and TFEBmt-treated samples may reflect the differences in the glycosylation pattern.C. Western blot of protein lysates from untreated, Ad-null, TFEB, and TFEBmt-treated myotubes with anti-caspase-3 antibody. No activated (cleaved) products are detected in any condition. α-Tubulin was used as a loading control.D. TUNEL assay shows the presence of some apoptotic cells in Ad-TFEB-infected cultures, but not in control cultures infected with adenovirus alone (n = 3). Bar: 10 µm for all panels.


Figure 4A . Confocal microscopy images of live fibres derived from 3 to 4 month - old GFP - LC3 : WT ( top left ) , untreated GFP - LC3 : GAA − / − ( bottom left ) or TFEB - treated GFP - LC3 : GAA − / − ( right ) mice . All fibres were transfected with mCherry - LAMP1 to visualize lysosomes ( red ) . The effects of TFEB are clearly visible - overall reduction in lysosomal size , appearance of normal size lysosomes ( similar to those in the WT ) , and lysosomal docking to the plasma membrane ( inset ) . Bar : 10 µm . B . FDBmuscle of a GAA − / − mouse was transfected with both GFP - TFEB and mCherry - LAMP1 ( GAA − / − + GFP - TFEB ) . Images were taken before ( left ) and after 4 h ( right ) of time - lapse microscopy . Lysosomal clearance is visible in the TFEB - transfected fibre at both time points . Lysosomes appear to " exit " the fibre ( inset and arrow ) at the 4 h time point when TFEB is activated as evidenced by its nuclear translocation ( green nuclei ) . Bar : 10 µm . C . The mean maximum velocity of lysosomes ( top ) and the number of large ( > 3 . 5 µm ) lysosomes ( bottom ) in untreated and TFEB - treated PD fibres ( note : all data for Flag - and GFP - TFEB - treated fibres are pooled ) . Lysosomal velocities were calculated from time - lapse images using ImageJ software with the manual tracking plug - in . For each condition the trajectories of multiple lysosomes were followed ( n = 26 untreated ; n = 43 TFEB - treated ) and the three highest velocity measurements per lysosome were recorded . In TFEB - treated fibres , the maximum velocity of lysosomes was significantly increased ( p = 2 . 07 × 10 − 17 ) and the number of large lysosomes was significantly decreased ( p = 1 . 0 × 10 − 3 ) . Ten untreated and 24 TFEB - treated fibres were analysed for the size calculations . * indicates statistically significant differences ( p ≤ 0 . 001 ; Student ' s t - test ) . Error bars represent 95 % confidence intervals .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Confocal microscopy images of live fibres derived from 3 to 4 month-old GFP-LC3:WT (top left), untreated GFP-LC3:GAA−/− (bottom left) or TFEB-treated GFP-LC3:GAA−/− (right) mice. All fibres were transfected with mCherry-LAMP1 to visualize lysosomes (red). The effects of TFEB are clearly visible - overall reduction in lysosomal size, appearance of normal size lysosomes (similar to those in the WT), and lysosomal docking to the plasma membrane (inset). Bar: 10 µm.B. FDBmuscle of a GAA−/− mouse was transfected with both GFP-TFEB and mCherry-LAMP1 (GAA−/− + GFP-TFEB). Images were taken before (left) and after 4 h (right) of time-lapse microscopy. Lysosomal clearance is visible in the TFEB-transfected fibre at both time points. Lysosomes appear to "exit" the fibre (inset and arrow) at the 4 h time point when TFEB is activated as evidenced by its nuclear translocation (green nuclei). Bar: 10 µm.C. The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated PD fibres (note: all data for Flag- and GFP-TFEB-treated fibres are pooled). Lysosomal velocities were calculated from time-lapse images using ImageJ software with the manual tracking plug-in. For each condition the trajectories of multiple lysosomes were followed (n = 26 untreated; n = 43 TFEB-treated) and the three highest velocity measurements per lysosome were recorded. In TFEB-treated fibres, the maximum velocity of lysosomes was significantly increased (p = 2.07 × 10−17) and the number of large lysosomes was significantly decreased (p = 1.0 × 10−3). Ten untreated and 24 TFEB-treated fibres were analysed for the size calculations. * indicates statistically significant differences (p ≤ 0.001; Student's t-test). Error bars represent 95% confidence intervals.


Figure 5Confocal microscopy images of live fibres from GFP - LC3 : GAA − / − mice . Muscle was transfected with mCherry - LAMP1 only . The image ( a single frame from the time - lapse series presented in Movie 5 ) shows lysosomes ( red ) , LC3 - positive autophagosomes ( green ) and a number of autolysosomes ( yellow ) . Muscle was transfected with Flag - TFEB and mCherry - LAMP1 . Massive formation of autolysosomes is indicated by yellow structures ; the three lower panels provide a snapshot of the process of exocytosis . The structures at the plasma membrane are labelled with both LC3 and LAMP1 , indicating that they represent amphisomes ( a product of fusion between autophagic vesicles and late endosomes ) or autolysosomes ( a product of fusion between autophagic vesicles and lysosomes ) . Bar : 10 µm for all panels .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Confocal microscopy images of live fibres from GFP-LC3:GAA−/− mice.Muscle was transfected with mCherry-LAMP1 only. The image (a single frame from the time-lapse series presented in Movie 5) shows lysosomes (red), LC3-positive autophagosomes (green) and a number of autolysosomes (yellow).Muscle was transfected with Flag-TFEB and mCherry-LAMP1. Massive formation of autolysosomes is indicated by yellow structures; the three lower panels provide a snapshot of the process of exocytosis. The structures at the plasma membrane are labelled with both LC3 and LAMP1, indicating that they represent amphisomes (a product of fusion between autophagic vesicles and late endosomes) or autolysosomes (a product of fusion between autophagic vesicles and lysosomes). Bar: 10 µm for all panels.


Figure 6Confocal microscopy images of live fibres from muscle - specific autophagy - deficient GAA − / − mice ( Atg7 : GAA DKO ) . Muscle was transfected with mCherry - LAMP1 only . Muscle was transfected with GFP - TFEB and mCherry - LAMP1 ( Atg7 : GAA DKO + GFP - TFEB ) . The TFEB - transfected fibres show realignment of the lysosomes and membrane detachment ( most striking in top and bottom panels ) similar to those in TFEB - transfected fibres from PD mice ( see Fig 4B inset ) . Lysosomes can be seen in the space between the fibre and plasma membrane ( arrows ) . Bar : 10 µm for all panels . The mean maximum velocity of lysosomes ( top ) and the number of large ( > 3 . 5 µm ) lysosomes ( bottom ) in untreated and TFEB - treated autophagy - deficient Atg7 : GAA DKO fibres . The increase in maximum velocity ( 41 % ) is significant ( p = 2 . 729 × 10 − 18 ; n = 57 lysosomes for untreated ; n = 52 lysosomes for TFEB - treated ) , but there is only a slight trend toward smaller lysosomal size ( p = 7 . 0 × 10 − 2 ; n = 10 fibres for untreated ; n = 21 fibres for TFEB - treated ) . The corresponding values from GAA − / − mice are presented for comparison ( for TFEB - treated condition : n = 19 lysosomes for velocity measurements , and n = 10 fibres for the lysosomal size measurements ) . * indicates statistically significant differences ( p ≤ 1 . 0 × 10 − 5 for the top panel and p ≤ 0 . 01 for the lower panel ; Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Confocal microscopy images of live fibres from muscle-specific autophagy-deficient GAA−/− mice (Atg7:GAA DKO).Muscle was transfected with mCherry-LAMP1 only.Muscle was transfected with GFP-TFEB and mCherry-LAMP1 (Atg7:GAA DKO +GFP-TFEB). The TFEB-transfected fibres show realignment of the lysosomes and membrane detachment (most striking in top and bottom panels) similar to those in TFEB-transfected fibres from PD mice (see Fig 4B inset). Lysosomes can be seen in the space between the fibre and plasma membrane (arrows). Bar: 10 µm for all panels.The mean maximum velocity of lysosomes (top) and the number of large (>3.5 µm) lysosomes (bottom) in untreated and TFEB-treated autophagy-deficient Atg7:GAA DKO fibres. The increase in maximum velocity (41%) is significant (p = 2.729 × 10−18; n = 57 lysosomes for untreated; n = 52 lysosomes for TFEB-treated), but there is only a slight trend toward smaller lysosomal size (p = 7.0 × 10−2; n = 10 fibres for untreated; n = 21 fibres for TFEB-treated). The corresponding values from GAA−/− mice are presented for comparison (for TFEB-treated condition: n = 19 lysosomes for velocity measurements, and n = 10 fibres for the lysosomal size measurements). * indicates statistically significant differences (p ≤ 1.0 × 10−5 for the top panel and p ≤ 0.01 for the lower panel; Student's t-test).


Figure 7A . Glycogen assay in TFEB - injected gastrocnemii and in the contralateral muscles . In TFEB - injected musclesglycogen levels were significantly decreased compared to those in untreated muscles . * indicates statistically significant differences ( p = 1 . 0 × 10 − 4 ; n = 6 ; Student ' s t - test ) . B . PAS staining of TFEB - treated muscle shows a reduction of lysosomalglycogen stores ( puncta ) compared to those in untreated muscle . Original magnification : 20 × . C . LAMP1 staining of TFEB - injected gastrocnemii and of the contralateral untreated muscles . In TFEB - treated muscles , the size of LAMP1 - positive vesicles was reduced . Bar : 2 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. Glycogen assay in TFEB-injected gastrocnemii and in the contralateral muscles. In TFEB-injected musclesglycogen levels were significantly decreased compared to those in untreated muscles. * indicates statistically significant differences (p = 1.0 × 10−4; n = 6; Student's t-test).B. PAS staining of TFEB-treated muscle shows a reduction of lysosomalglycogen stores (puncta) compared to those in untreated muscle. Original magnification: 20×.C. LAMP1 staining of TFEB-injected gastrocnemii and of the contralateral untreated muscles. In TFEB-treated muscles, the size of LAMP1-positive vesicles was reduced. Bar: 2 µm.


Figure 8A . EM images of muscle injected with either AAV - GFP ( untreated ) or AAV - TFEB ( TFEB - treated ) . Asterisks indicate glycogen - containing lysosomes . Bar : 1 . 5 µm ( upper panels ) and 0 . 45 µm ( lower panels ) . Higher magnification images ( lower panels ) show that glycogen particles are less densely packed in TFEB - treated muscle . Black arrows indicate autophagosome profiles ; the white empty arrow shows remnants of mitochondria engulfed by the lysosome . B . Graphical presentations of lysosomal length ( average ± SE ; n = 100 lysosomes ; p = 6 . 31 × 10 − 5 ) , the number of lysosomes per 5 µm2 area of muscle fibre section ( average ± SE ; n = 50 fields ; p = 4 . 80 × 10 − 3 ) , and the number of autophagosomes flanking glycogen - containing lysosomes ( average ± SE ; n = 100 lysosomes ; p = 4 . 39 × 10 − 5 < 0 . 001 ) . Student ' s t - test was used for each comparison . C . Graphical presentations of mitochondrial size ( average ± SE ; n = 100 ) and the number of mitochondria per 5 µm2 area of muscle fibre section ( average ± SE ; n = 50 fields ) . The differences were not significant by Student ' s t - test ( p = 3 . 65 × 10 − 1 and 4 . 27 × 10 − 1 , respectively ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8A. EM images of muscle injected with either AAV-GFP (untreated) or AAV-TFEB (TFEB-treated). Asterisks indicate glycogen-containing lysosomes. Bar: 1.5 µm (upper panels) and 0.45 µm (lower panels). Higher magnification images (lower panels) show that glycogen particles are less densely packed in TFEB-treated muscle. Black arrows indicate autophagosome profiles; the white empty arrow shows remnants of mitochondria engulfed by the lysosome.B. Graphical presentations of lysosomal length (average ± SE; n = 100 lysosomes; p = 6.31 × 10−5), the number of lysosomes per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields; p = 4.80 × 10−3), and the number of autophagosomes flanking glycogen-containing lysosomes (average ± SE; n = 100 lysosomes; p = 4.39 × 10−5 < 0.001). Student's t-test was used for each comparison.C. Graphical presentations of mitochondrial size (average ± SE; n = 100) and the number of mitochondria per 5 µm2 area of muscle fibre section (average ± SE; n = 50 fields). The differences were not significant by Student's t-test (p = 3.65 × 10−1 and 4.27 × 10−1, respectively).


Figure 1A Phenotypes of WT ( Col - 0 ) , two gso1 mutant alleles ( gso1 - 1 , gso1 - 3 ) and two independent complementation lines ( com - 1 , com - 2 ) . Seedlings were grown on 1 / 2 MS for 6 days and then transferred to 1 / 2 MS medium supplemented with or without 100 mM NaCl for 10 days . B , C Primary root length and seedling fresh weight of seedlings depicted in ( A ) were measured at day 10 after transfer . ( mean ± SEM , n = 10 , P < 0 . 05 , two - way ANOVA )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A Phenotypes of WT (Col-0), two gso1 mutant alleles (gso1-1, gso1-3) and two independent complementation lines (com-1, com-2). Seedlings were grown on 1/2 MS for 6 days and then transferred to 1/2 MS medium supplemented with or without 100 mM NaCl for 10 days. B,C Primary root length and seedling fresh weight of seedlings depicted in (A) were measured at day 10 after transfer. (mean ± SEM, n = 10, P < 0.05, two-way ANOVA)


Figure 2A Co - IP of GSO1 and SOS2 using Arabidopsis protoplasts transiently expressing Myc - GSO1KD ( GSO1 kinase domain ) and Flag - SOS2 , Flag - HA - SOS3 or Flag - SCaBP8 . Protein extracts were immunoprecipitated with anti - Myc antibody and immunoblotted with anti - Myc antibody or anti - Flag antibody . C High resolution localization analyses of SOS2 and GSO1 expressed under their respective native promoter at 1 . 5 mm from the quiescent center . Propidium iodide ( PI ) was used to stain the cell wall . Scale bars : 100 µm . ( BF : brightfield ) D Phenotypes of WT ( Col - 0 ) , gso1 - 3 , sos2 - 2 and gso1 - 3 sos2 - 2 . Seedlings were grown on 1 / 2 MS medium for 6 days and then transferred to 1 / 2 MS supplemented with or without extra NaCl for 10 days . E , F Primary root length and fresh weight of seedlings depicted in ( D ) were measured at day 7 after transfer . ( mean ± SEM , n = 10 , P < 0 . 05 , two - way ANOVA )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A Co-IP of GSO1 and SOS2 using Arabidopsis protoplasts transiently expressing Myc-GSO1KD (GSO1 kinase domain) and Flag-SOS2, Flag-HA-SOS3 or Flag-SCaBP8. Protein extracts were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-Myc antibody or anti-Flag antibody.C High resolution localization analyses of SOS2 and GSO1 expressed under their respective native promoter at 1.5 mm from the quiescent center. Propidium iodide (PI) was used to stain the cell wall. Scale bars: 100 µm. (BF: brightfield)D Phenotypes of WT (Col-0), gso1-3, sos2-2 and gso1-3 sos2-2. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS supplemented with or without extra NaCl for 10 days. E,F Primary root length and fresh weight of seedlings depicted in (D) were measured at day 7 after transfer. (mean ± SEM, n = 10, P < 0.05, two-way ANOVA)


Figure 3A Yeast strain JP837 cells transformed with empty vectors ( control ) or the indicated combinations of Arabidopsis genes were grown on AP medium supplemented with or without 100 mM NaCl for 3 days at 28 ℃ . ( M : myristoylation motif , KD : kinase domain ) G - L Phenotypes , primary root length and seedling fresh weight of indicated plant lines grown on 1 / 2 MS medium for 6 days and then transferred to 1 / 2 MS medium supplemented with 100 mM NaCl for 7 days . ( mean ± SEM , n = 10 , P < 0 . 05 , one - way ANOVA )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Yeast strain JP837 cells transformed with empty vectors (control) or the indicated combinations of Arabidopsis genes were grown on AP medium supplemented with or without 100 mM NaCl for 3 days at 28℃. (M: myristoylation motif, KD: kinase domain)G-L Phenotypes, primary root length and seedling fresh weight of indicated plant lines grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with 100 mM NaCl for 7 days. (mean ± SEM, n = 10, P < 0.05, one-way ANOVA)


Figure 4A Coomassie Brilliant Blue stain ( CBB ) and autoradiograph of in vitro kinase assay combining the indicated combinations of SOS2 / SOS2K40N and GSO1KD / GSO1KDK979N . B Immunoblot using anti - Myc antibody and CBB and autoradiograph from kinase assays combining Myc - SOS2 with SOS1C300 as substrate . 10 - day - old seedlings stably expressing Pro35S : 6Myc - SOS2 in Col - 0 or gso1 - 3 were treated with or without 100 mM NaCl for 12 h , and Myc - SOS2 protein immunoprecipitated with anti - C - Myc antibody - conjugated agarose from roots was used in the assays . Signal strength of the phosphorylation bands has been calculated relative to the left lane . C CBB and autoradiograph of in vitro kinase assays combining GSO1KD ( kinase domain ) with SOS2K40N or SOS2T16AK40N . D CBB and autoradiograph of in vitro kinase assays using SOS2 p - site variants addressing autophosphorylation activity .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Coomassie Brilliant Blue stain (CBB) and autoradiograph of in vitro kinase assay combining the indicated combinations of SOS2/SOS2K40N and GSO1KD/GSO1KDK979N.B Immunoblot using anti-Myc antibody and CBB and autoradiograph from kinase assays combining Myc-SOS2 with SOS1C300 as substrate. 10-day-old seedlings stably expressing Pro35S:6Myc-SOS2 in Col-0 or gso1-3 were treated with or without 100 mM NaCl for 12 h, and Myc-SOS2 protein immunoprecipitated with anti-C-Myc antibody-conjugated agarose from roots was used in the assays. Signal strength of the phosphorylation bands has been calculated relative to the left lane.C CBB and autoradiograph of in vitro kinase assays combining GSO1KD (kinase domain) with SOS2K40N or SOS2T16AK40N. D CBB and autoradiograph of in vitro kinase assays using SOS2 p-site variants addressing autophosphorylation activity.


Figure 5A Phenotypes of WT ( Col - 0 ) , gso1 - 3 , esb1 , and casp1 casp3 . Seedlings were grown on 1 / 2 MS for 6 days and then transferred to 1 / 2 MS medium with or without 100 mM NaCl for 10 days . B , C Primary root length and seedling fresh weight of seedlings depicted in ( A ) were measured at day 10 after transfer . ( mean ± SEM , n = 12 , two - way ANOVA , P < 0 . 05 ) G Phenotypes of WT ( Col - 0 ) , cif1 cif2 , and gso1 - 3 . Seedlings were grown on 1 / 2 MS medium for 6 days and then transferred to 1 / 2 MS medium supplemented with or without 100 mM NaCl for 10 days . J Quantification of Fluorol Yellow fluorescence intensity indicating amounts of suberin accumulation in maximum projections of z - stacks in the first mm of the continuously suberized endodermis in the indicated genotypes . Seedlings were grown on 1 / 2 MS for 5 days and then treated with the indicated NaCl concentrations for 2 days . ( mean ± SEM , n = 3 - 5 , P < 0 . 05 , one - way ANOVA and post hoc Tukey Kramer test , different letters indicate significant difference )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Phenotypes of WT (Col-0), gso1-3, esb1, and casp1 casp3. Seedlings were grown on 1/2 MS for 6 days and then transferred to 1/2 MS medium with or without 100 mM NaCl for 10 days. B, C Primary root length and seedling fresh weight of seedlings depicted in (A) were measured at day 10 after transfer. (mean ± SEM, n = 12, two-way ANOVA, P < 0.05)G Phenotypes of WT (Col-0), cif1 cif2, and gso1-3. Seedlings were grown on 1/2 MS medium for 6 days and then transferred to 1/2 MS medium supplemented with or without 100 mM NaCl for 10 days.J Quantification of Fluorol Yellow fluorescence intensity indicating amounts of suberin accumulation in maximum projections of z-stacks in the first mm of the continuously suberized endodermis in the indicated genotypes. Seedlings were grown on 1/2 MS for 5 days and then treated with the indicated NaCl concentrations for 2 days. (mean ± SEM, n = 3-5, P < 0.05, one-way ANOVA and post hoc Tukey Kramer test, different letters indicate significant difference)


Figure 6A - D Protein accumulation ( A , C , D ) or gene expression ( B ) of SOS2 , CIF2 , GSO1 , and SGN1 displayed as false color fluorescence intensity . Seedlings from the indicated plant lines were grown on 1 / 2 MS for 4 - 5 days and then transferred to 1 / 2 MS with or without 100 mM NaCl for 1 day . Scale bars : 200 µm . E - H Longitudinal quantification of protein accumulation or gene expression depicted in ( A - D ) represented as 8 - bit fluorescence intensity . ( Mean ± SEM , n = 4 - 5 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A-D Protein accumulation (A, C, D) or gene expression (B) of SOS2, CIF2, GSO1, and SGN1 displayed as false color fluorescence intensity. Seedlings from the indicated plant lines were grown on 1/2 MS for 4-5 days and then transferred to 1/2 MS with or without 100 mM NaCl for 1 day. Scale bars: 200 µm.E-H Longitudinal quantification of protein accumulation or gene expression depicted in (A-D) represented as 8-bit fluorescence intensity. (Mean ± SEM, n = 4-5)


Figure 1 ( A ) In vivo fundusimage of an AAV - injected rd1mouse expressing ReaChR - mCitrine driven by the hSyn promoter ( AAV2 - hSyn : ReaChR - mCitrine ) ; transduced RGCs are visible as green puncta . ( B ) Live 2 - photon image showing that ReaChR - mCitrine expression is restricted to RGC - membranes and their dendritic processes . ( C ) Confocal image of a vertical retinal section expressing ReaChR - mCitrine . Endogenous mCitrine ( mCit ) fluorescence , is localized to RGCmembranes as well as RGCdendritic processes ( see also Fig EV1A ) ; blue : DAPI ( to visualize retinalnuclei ) ; inner nuclear layer ( INL ) ; inner plexiform layer ( IPL ) and ganglion cell layer ( GCL ) ; scale bars = 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) In vivo fundusimage of an AAV-injected rd1mouse expressing ReaChR-mCitrine driven by the hSyn promoter (AAV2-hSyn:ReaChR-mCitrine); transduced RGCs are visible as green puncta.(B) Live 2-photon image showing that ReaChR-mCitrine expression is restricted to RGC-membranes and their dendritic processes.(C) Confocal image of a vertical retinal section expressing ReaChR-mCitrine. Endogenous mCitrine (mCit) fluorescence, is localized to RGCmembranes as well as RGCdendritic processes (see also Fig EV1A); blue: DAPI (to visualize retinalnuclei); inner nuclear layer (INL); inner plexiform layer (IPL) and ganglion cell layer (GCL); scale bars = 10 µm.


Figure 2 ( A ) Light sensitivity : Photocurrents of a ReaChR - expressing RGC stimulated with varying light intensities at 590 nm ( ~ 1013 − 1018 photons cm - 2 s - 1 ) . ( B ) ReaChR - induced photocurrents as a function of light intensity ( 590 nm , n = 7 cells , mean photocurrent amplitude at 4 . 2 × 1017 photons cm - 2 s - 1 was − 349 ± 192 pA ) . At the bottom , the sensitivity range for wild - type cones ( Nikonov et al , 2006 ) is shown in comparison to the sensitivity range of ReaChR expressing RGCs . The dashed blue and orange line indicate the maximum light intensities allowed in the human eye at 470nm and 590 nm , respectively ( EU - Directive , 2006 ; ICNIRP , 2013 ) . ( C ) Photocurrent action spectrum of a ReaChR - expressing RGC ( 1 . 2 × 1016 photons cm - 2 s - 1 ) . ( D ) Normalized action spectrums of ReaChR - RGCs . Solid line represents patch - clamp data ( n = 8 cells ; 1 . 2 × 1016 photons cm - 2 s - 1 ) . The mean photocurrent amplitude at 550 nm was − 324 ± 97 pA . Dashed line represents multi - electrode array data ( n = 168 cells ; ~ 1016 photons cm - 2 s - 1 ) . ( E ) Temporal properties . Modulation of ReaChR - induced photocurrents at increasing stimulation frequencies ( patch - clamp recording from a ReaChR - expressing RGC ; at 22 Hz magnified trace is shown ; 1 . 3 × 1016 photons cm - 2 s - 1 ) . ( F ) Spiking modulation ratio of ReaChR - RGCs quantified from multi - electrode array demonstrates reliable spike triggering at frequencies up to 30 Hz ( 297 cells , 1 . 3 × 1016 photons cm - 2 s - 1 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Light sensitivity: Photocurrents of a ReaChR-expressing RGC stimulated with varying light intensities at 590 nm (~1013 − 1018 photons cm-2 s-1).(B) ReaChR-induced photocurrents as a function of light intensity (590 nm, n = 7 cells, mean photocurrent amplitude at 4.2 × 1017 photons cm-2 s-1 was −349 ± 192 pA). At the bottom, the sensitivity range for wild-type cones (Nikonov et al, 2006) is shown in comparison to the sensitivity range of ReaChR expressing RGCs. The dashed blue and orange line indicate the maximum light intensities allowed in the human eye at 470nm and 590 nm, respectively (EU-Directive, 2006; ICNIRP, 2013).(C) Photocurrent action spectrum of a ReaChR-expressing RGC (1.2 × 1016 photons cm-2 s-1). (D) Normalized action spectrums of ReaChR-RGCs. Solid line represents patch-clamp data (n = 8 cells; 1.2 × 1016 photons cm-2 s-1). The mean photocurrent amplitude at 550 nm was −324 ± 97 pA. Dashed line represents multi-electrode array data (n = 168 cells; ~1016 photons cm-2 s-1).(E) Temporal properties. Modulation of ReaChR-induced photocurrents at increasing stimulation frequencies (patch-clamp recording from a ReaChR-expressing RGC; at 22 Hz magnified trace is shown; 1.3×1016 photons cm-2 s-1). (F) Spiking modulation ratio of ReaChR-RGCs quantified from multi-electrode array demonstrates reliable spike triggering at frequencies up to 30 Hz (297 cells, 1.3 × 1016 photons cm-2 s-1).


Figure 3 ( A ) Examples of local field potential ( LFP ) traces in response to light flashes at 595 nm ( light stimulus is shown as orange bar ; 1 . 25 × 1017 photons cm - 2 s - 1 ) . ( B ) Spike raster plots in response to 200 repetitions of the same light stimulus . ( C ) Averaged spike responses shown as peristimulus time histograms ( PSTH , bin size : 5 ms ) based on the spike raster plots . Top - row : WT - mice , middle row : rd1 control mice , bottom - row : ReaChR - treated rd1 mice . ( D ) ReaChR - induced cortical responses ( spikes and LFP ) as a function of light intensity at ( 595 nm ) demonstrating that ReaChR - treated rd1 mice ( n = 4 ) can detect light in the visual cortex at intensities well below the safety threshold allowed for the humanretina . Data presented as mean ± SEM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Examples of local field potential (LFP) traces in response to light flashes at 595 nm (light stimulus is shown as orange bar; 1.25 × 1017 photons cm-2 s-1). (B) Spike raster plots in response to 200 repetitions of the same light stimulus. (C) Averaged spike responses shown as peristimulus time histograms (PSTH, bin size: 5 ms) based on the spike raster plots. Top-row: WT-mice, middle row: rd1 control mice, bottom-row: ReaChR-treated rd1 mice. (D) ReaChR-induced cortical responses (spikes and LFP) as a function of light intensity at (595 nm) demonstrating that ReaChR-treated rd1 mice (n = 4) can detect light in the visual cortex at intensities well below the safety threshold allowed for the humanretina. Data presented as mean ± SEM.


Figure 4 ( A ) Locomotory behavior of a representative rd1 and ReaChR - treated mouse during illumination with light at 590 nm in an open field arena ( irradiance measured at the level of the mice ' s eyes was ~ 1015 photons cm - 2 s - 1 , cumulative activity trajectories are shown as red traces after 2 min of illumination ) , see also EV Movie 1 and EV Movie 2 . ( B ) Mean velocities of the ReaChR - treated and rd1mice analyzed over a duration of 2 min at light onset . The mean velocity of ReaChR - treated mice were significantly reduced than rd1mice ( P = 0 . 006 , one - tailed t - test ) upon illumination . Mean ± SEM are shown for each group . ReaChR - treated rd1 ( n = 11 ) , rd1 ( n = 12 ) . ( C ) Light / dark box test . Percentage of time mice spent in the light compartment as a function of light intensity ( WT , n = 9 ; rd1 , n = 9 ; ReaChR - treated n = 7 ) . Mean ± SEM are shown for each group . ( D ) Percentage of time rd1 ( n = 9 ) , WT ( n = 9 ) and ReaChR - treated ( n = 7 ) mice spent in the light compartment for an illumination intensity of 2 . 11 × 1015 photons cm - 2 s - 1 . Both WT as well as ReaChR - treated mice spent significantly less time in the light compartment than the rd1mice ( P < 0 . 0001 , ordinary one - way ANOVA ) . * * * and * * * * indicates significance levels determined by Tukey ' s multiple comparisons test for rd1 vs . WT and rd1 vs . ReaChR - treated . See also EV Movie 3 for the response of a representative ReaChR - treated rd1 mouse plotted in ( D ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Locomotory behavior of a representative rd1 and ReaChR-treated mouse during illumination with light at 590 nm in an open field arena (irradiance measured at the level of the mice's eyes was ~1015 photons cm-2 s-1, cumulative activity trajectories are shown as red traces after 2 min of illumination), see also EV Movie 1 and EV Movie 2. (B) Mean velocities of the ReaChR-treated and rd1mice analyzed over a duration of 2 min at light onset. The mean velocity of ReaChR-treated mice were significantly reduced than rd1mice (P = 0.006, one-tailed t-test) upon illumination. Mean ± SEM are shown for each group. ReaChR-treated rd1 (n = 11), rd1 (n = 12).(C) Light/dark box test. Percentage of time mice spent in the light compartment as a function of light intensity (WT, n = 9; rd1, n = 9; ReaChR-treated n = 7). Mean ± SEM are shown for each group. (D) Percentage of time rd1 (n = 9), WT (n = 9) and ReaChR-treated (n = 7) mice spent in the light compartment for an illumination intensity of 2.11 × 1015 photons cm-2 s-1. Both WT as well as ReaChR-treated mice spent significantly less time in the light compartment than the rd1mice (P < 0.0001, ordinary one-way ANOVA). *** and **** indicates significance levels determined by Tukey's multiple comparisons test for rd1 vs. WT and rd1 vs. ReaChR-treated. See also EV Movie 3 for the response of a representative ReaChR-treated rd1 mouse plotted in (D).


Figure 5 ( A ) Vertical section of macaqueretinal explant transfected with AAV2 hSyn : ReaChR - mCit after 10 days of incubation exhibiting mCitrine expresssion densely in neuronal processes in the GCL . ( B ) Vertical section of macaqueretinal explant transfected with AAV8 Y447 733F - CAG : ReaChR - GFP after 3 days of incubation . GFP expression was primarily observed in the cell bodies but also in processes of RGCs ; green : endogenous GFP fluorescence ; blue : DAPI ; outer nuclear layer ( ONL ) . scale bars = 10 µm . ( C ) Averaged spike responses obtained from multi - electrode array recordings shown as PSTH of 4 , 8 and 2 cells respectively of three different macaques ( 580 nm ; 1 . 24 × 1017 photons cm - 2 s - 1 ) . PSTHs are presented as mean ± SEM ( dashed lines , calculated over responding cells ) . Raster plots of a representative cell from each individual macaque are shown below ( 10 stimulus - repetitions ) . ( D ) Action spectrum of ReaChR in primate RGCs from multi - electrode array recordings . Data is shown as mean ± SEM of the normalized firing rate , averaged from three individual macaques ( 28 cells , error bars calculated over cells ) , see also Fig EV2B for action spectrums of three individual macaques obtained from multi - electrode array recordings . ( E ) Responses ( Hz ) of a ReaChR - expressing RGC ( patch clamp recording , in cell - attached mode ) to flicker stimulations at increasing frequencies ( 2 − 22 Hz ; 1 . 3 × 1016 photons cm - 2 s - 1 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Vertical section of macaqueretinal explant transfected with AAV2 hSyn:ReaChR-mCit after 10 days of incubation exhibiting mCitrine expresssion densely in neuronal processes in the GCL. (B) Vertical section of macaqueretinal explant transfected with AAV8 Y447 733F-CAG:ReaChR-GFP after 3 days of incubation. GFP expression was primarily observed in the cell bodies but also in processes of RGCs; green: endogenous GFP fluorescence; blue: DAPI; outer nuclear layer (ONL). scale bars = 10 µm.(C) Averaged spike responses obtained from multi-electrode array recordings shown as PSTH of 4, 8 and 2 cells respectively of three different macaques (580 nm; 1.24 × 1017 photons cm-2 s-1). PSTHs are presented as mean ± SEM (dashed lines, calculated over responding cells). Raster plots of a representative cell from each individual macaque are shown below (10 stimulus-repetitions).(D) Action spectrum of ReaChR in primate RGCs from multi-electrode array recordings. Data is shown as mean ± SEM of the normalized firing rate, averaged from three individual macaques (28 cells, error bars calculated over cells), see also Fig EV2B for action spectrums of three individual macaques obtained from multi-electrode array recordings. (E) Responses (Hz) of a ReaChR-expressing RGC (patch clamp recording, in cell-attached mode) to flicker stimulations at increasing frequencies (2−22 Hz; 1.3 × 1016 photons cm-2 s-1).


Figure 6 ( B ) Brightfield image of the retinal explant showing the macula ( yellow pigmented ) with the approximate location of the multi - electrode array chip indicated . ( C ) Infrared image of the explant pressed against the multi - electrode array ; the black dashed line depicts the ' electrophysiological boundary ' of the para - fovea based on the absence of RGC - spike trains ( scale bar : 100 µm ; see also Fig EV3A and B ) . Voltage trace of a para - foveal RGC stimulated with orange light ( 600 nm ; 1 . 1 × 1017 photons cm - 2 s - 1 ) is shown on the right . Individual electrodes that recorded ReaChR - responding cells are indicated in green . ( D ) Action spectrum of ReaChR constructed from ( 6 cells , mean ± SEM , error bars calculated over cells ) exhibiting peak activity at 550 nm ( 1 . 1 × 1017 photons cm - 2 s - 1 ) similar to what was observed in mice and in the macaqueretinae . ( E ) Peristimulus time histograms constructed for wavelengths 400 to 650 nm ( 1 . 1 × 1017 photons cm - 2 s - 1 ) demonstrate ReaChR - evoked spike trains ( under L - AP4 ) in the humanretina ( 6 cells , mean ± SEM , error bars calculated over cells ) , bin size : 100 ms ; Bottom : Raster plots of the light responses of a representative spike - sorted cell .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(B) Brightfield image of the retinal explant showing the macula (yellow pigmented) with the approximate location of the multi-electrode array chip indicated.(C) Infrared image of the explant pressed against the multi-electrode array; the black dashed line depicts the 'electrophysiological boundary' of the para-fovea based on the absence of RGC-spike trains (scale bar: 100 µm; see also Fig EV3A and B). Voltage trace of a para-foveal RGC stimulated with orange light (600 nm; 1.1 × 1017 photons cm-2 s-1) is shown on the right. Individual electrodes that recorded ReaChR-responding cells are indicated in green.(D) Action spectrum of ReaChR constructed from (6 cells, mean ± SEM, error bars calculated over cells) exhibiting peak activity at 550 nm (1.1 × 1017 photons cm-2 s-1) similar to what was observed in mice and in the macaqueretinae.(E) Peristimulus time histograms constructed for wavelengths 400 to 650 nm (1.1 × 1017 photons cm-2 s-1) demonstrate ReaChR-evoked spike trains (under L-AP4) in the humanretina (6 cells, mean ± SEM, error bars calculated over cells), bin size: 100 ms; Bottom: Raster plots of the light responses of a representative spike-sorted cell.


Figure 7 ( A ) Confocal image of the central retina , exhibiting endogenous ReaChR - mCitrine fluorescence ( mCit ) in ganglion cells , located within ~ 500 µm from the center of the foveal pit , a part of the humanretina where midget cell density is estimated to be ~ 95 % of total RGC population ( Dacey , 1993 ) ( arrowhead : soma ; arrow : dendritic arbor ) . The dashed line illustrates the beginning of the foveal slope leading towards the ' cone - only ' foveal pit . ( B ) Confocal image of the same explant at a higher retinal eccentricity ( 3 - 4 mm from the foveal center ) showing ReaChR - mCitrine fluorescence in RGCs with larger dendritic arbors . The endogenous mCitrine was not amplified . Scale bars : 10 µm . PR : Photoreceptors , GC : Ganglion cells , DAPI ( blue ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Confocal image of the central retina, exhibiting endogenous ReaChR-mCitrine fluorescence (mCit) in ganglion cells, located within ~500 µm from the center of the foveal pit, a part of the humanretina where midget cell density is estimated to be ~95% of total RGC population (Dacey, 1993) (arrowhead: soma; arrow: dendritic arbor). The dashed line illustrates the beginning of the foveal slope leading towards the 'cone-only' foveal pit. (B) Confocal image of the same explant at a higher retinal eccentricity (3-4 mm from the foveal center) showing ReaChR-mCitrine fluorescence in RGCs with larger dendritic arbors. The endogenous mCitrine was not amplified. Scale bars: 10 µm. PR: Photoreceptors, GC: Ganglion cells, DAPI (blue).