Figure 6Combination gene therapy with IFNα , TNFα and IFNγ , expressed from the Tie2 vector platform . CTRL ( Tie2 . NGFR ) , 2 independent experiments , n = 9 mice ; IFNα , 1 experiment , n = 2 mice ; IFNα / IFNγ , 1 experiment , n = 4 mice ; IFNα / TNFα , 1 experiment , n = 3 mice ; IFNγ / TNFα , 2 independent experiments , n = 19 mice . ( C ) Percentage of MHC II + macrophages identified by F4 / 80 expression in the spleen ( mean ± SD , each dot represents an individual mouse ; * p = 0 . 0484 , * * p = 0 . 0049 , ordinary One - Way ANOVA ) . ( D ) Percentage of CD8 + T lymphocytes within CD45 positive and OFP negative splenic cells ( mean ± SD , each dot represents an individual mouse ; * p = 0 . 0356 , ordinary One - Way ANOVA ) . ( Combination of IFNγ gene therapy with immuno - oncology drugs . CTRL ( Tie2 . NGFR ) + antibody isotype , n = 6 mice ; CTRL + αCTLA4 antibody , n = 5 mice ; CTRL + αLAG3 antibody , n = 5 mice ; CTRL + 1 - Methyltryptophan ( 1 - MT ) , n = 5 mice ; IFNγ ( Tie2 . IFNγ ) + antibody isotype , n = 7 mice ; IFNγ + αCTLA4 antibody , n = 7 mice ; IFNγ + αLAG3 antibody , n = 7 mice ; IFNγ + 1 - MT , n = 5 mice . ( F ) Percentage of MHC II + macrophages identified by F4 / 80 expression in the spleen ( mean ± SD , each dot represents an individual mouse ; * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 , * * * * p ≤ 0 . 0001 as compared to CTRL isotype , ordinary One - Way ANOVA ) . ( G ) Percentage of CD8 + T lymphocytes within OFP negative BM cells ( mean ± SD , each dot represents an individual mouse ) . S

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Figure 6Combination gene therapy with IFNα, TNFα and IFNγ, expressed from the Tie2 vector platform. CTRL (Tie2.NGFR), 2 independent experiments, n = 9 mice; IFNα, 1 experiment, n = 2 mice; IFNα/IFNγ, 1 experiment, n= 4 mice; IFNα/TNFα, 1 experiment, n = 3 mice; IFNγ/TNFα, 2 independent experiments, n = 19 mice. (C) Percentage of MHC II+ macrophages identified by F4/80 expression in the spleen (mean ± SD, each dot represents an individual mouse; *p = 0.0484, **p = 0.0049, ordinary One-Way ANOVA). (D) Percentage of CD8+ T lymphocytes within CD45 positive and OFP negative splenic cells (mean ± SD, each dot represents an individual mouse; *p = 0.0356, ordinary One-Way ANOVA). ( Combination of IFNγ gene therapy with immuno-oncology drugs. CTRL (Tie2.NGFR) + antibody isotype, n = 6 mice; CTRL + αCTLA4 antibody, n= 5 mice; CTRL + αLAG3 antibody, n = 5 mice; CTRL + 1-Methyltryptophan (1-MT), n = 5 mice; IFNγ (Tie2.IFNγ) + antibody isotype, n = 7 mice; IFNγ + αCTLA4 antibody, n = 7 mice; IFNγ + αLAG3 antibody, n = 7 mice; IFNγ +1-MT, n = 5 mice. (F) Percentage of MHC II+ macrophages identified by F4/80 expression in the spleen (mean ± SD, each dot represents an individual mouse; **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001 as compared to CTRL isotype, ordinary One-Way ANOVA). (G) Percentage of CD8+ T lymphocytes within OFP negative BM cells (mean ± SD, each dot represents an individual mouse). S


Figure 3 ( A ) Representative traces showing SQR - driven oxygen consumption in 3x106 control ( C ) and five patientsfibroblasts ( P1 - 5 ) in the absence ( solid traces ) or presence ( dashed traces ) of CoQ10 in the culture medium . See Materials and Methods for the role and concentration of the compounds used . ( B ) Relative rates of SQR - driven oxygen consumption are normalized to CIV respiration rates .

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Figure 3(A) Representative traces showing SQR-driven oxygen consumption in 3x106 control (C) and five patientsfibroblasts (P1-5) in the absence (solid traces) or presence (dashed traces) of CoQ10 in the culture medium. See Materials and Methods for the role and concentration of the compounds used. (B) Relative rates of SQR-driven oxygen consumption are normalized to CIV respiration rates.


Figure 4 ( A ) Representative western blot showing the level of SQR , TST , ETHE1 and SUOX proteins in three control ( C1 - 3 ) and four patients fibroblasts ( P1 - 4 ) . ( B - E ) Relative levels of proteins normalized to Vinculin .

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Figure 4(A) Representative western blot showing the level of SQR, TST, ETHE1 and SUOX proteins in three control (C1-3) and four patients fibroblasts (P1-4). (B-E) Relative levels of proteins normalized to Vinculin.


Figure 5 ( A ) Representative western blots showing the level of SQR , TST , ETHE1 and SUOX proteins in two controls ( C1 , C2 ) and two patients fibroblasts ( P3 , P4 ) . ( B - E ) Relative levels of proteins normalized to Vinculin .

Figure 5(A) Representative western blots showing the level of SQR, TST, ETHE1 and SUOX proteins in two controls (C1, C2) and two patients fibroblasts (P3, P4). (B-E) Relative levels of proteins normalized to Vinculin.


Figure 6 ( A ) Representative western blots showing SQR , TST , ETHE1 and SUOX of two control fibroblasts treated with DMSO ( - ) or with DMSO + 4 - NB ( + ) . ( B ) Quantification of proteins levels in fibroblasts treated with 4 - NB relative to control . ( C ) Representative western blots showing ADCK3 , SQR , TST , ETHE1 and SUOX in two control ( EV ) and six ADCK3 - depleted clones ( ADCK3 RNAi ) . ( D ) Quantification of proteins levels in ADCK3 - depleted clones ( ADCK3 RNAi ) relative to controls ( EV ) .

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Figure 6(A) Representative western blots showing SQR, TST, ETHE1 and SUOX of two control fibroblasts treated with DMSO (-) or with DMSO+ 4-NB (+). (B) Quantification of proteins levels in fibroblasts treated with 4-NB relative to control.(C) Representative western blots showing ADCK3, SQR, TST, ETHE1 and SUOX in two control (EV) and six ADCK3-depleted clones (ADCK3 RNAi). (D) Quantification of proteins levels in ADCK3-depleted clones (ADCK3 RNAi) relative to controls (EV).


Figure 7Distribution of peptides containing sulfhydrated cysteine residues relative to their H / L ratios as determined by the BTA analysis of cell extracts isolated from C1 and P1 patient cells ( indicated in A ) and C2 and P4 fibroblasts ( B ) . Values of H / L ratios are plotted against the number of identified peptides . The gray line marks the H / L ratio > 2 , consisting of cysteine - containing peptides in proteins from patient cell lines that exhibited higher reactivity with H2S compared with the respective control . Indicated on the X axes are representative sensitive cysteine peptides , which are common between the ones found in the RedoxDB database , and by the BTA assay .

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Figure 7Distribution of peptides containing sulfhydrated cysteine residues relative to their H/L ratios as determined by the BTA analysis of cell extracts isolated from C1 and P1 patient cells (indicated in A) and C2 and P4 fibroblasts (B). Values of H/L ratios are plotted against the number of identified peptides. The gray line marks the H/L ratio >2, consisting of cysteine-containing peptides in proteins from patient cell lines that exhibited higher reactivity with H2S compared with the respective control. Indicated on the X axes are representative sensitive cysteine peptides, which are common between the ones found in the RedoxDB database, and by the BTA assay.


Figure 8 ( A ) Representative western blots showing the levels of SQR , TST , ETHE1 and SUOX protein in the kidney of three wild - type ( WT ) and three mutant ( Mut ) mice . ( B - E ) Relative levels of proteins normalized to Vinculin .

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Figure 8(A) Representative western blots showing the levels of SQR, TST, ETHE1 and SUOX protein in the kidney of three wild-type (WT) and three mutant (Mut) mice. (B-E) Relative levels of proteins normalized to Vinculin.


Figure 9 ( A ) Representative western blots showing the levels of SQR , TST , ETHE1 and SUOX protein from the brain of 5 wild - type ( WT ) and five mutant ( Mut ) mice ( B - E ) Relative levels of proteins normalized to Vinculin .

Figure 9(A) Representative western blots showing the levels of SQR, TST, ETHE1 and SUOX protein from the brain of 5 wild-type (WT) and five mutant (Mut) mice (B-E) Relative levels of proteins normalized to Vinculin.


Figure 10 ( A ) Total GSH amount normalized to protein from kidney and ( B ) brain of wild - type ( WT ) and mutant ( Mut ) mice . Error bars represent SDs of at least 3 animals per group . ( C ) Thiosulfate levels in urine and ( D ) plasma normalized to protein amount . Error bars represent SDs of 9 animals per group ( urine ) and 6 animals per group ( plasma ) . ( E ) H2S amount normalized to tissue from kidney and ( F ) brain . Error bars represent SDs of 7 / 8 animals per group . ( G ) Acylcarnitine C4 , C5 and C6 levels from plasma , normalized to volume . Error bars represent SDs of 5 wild - type and 3 mutants .

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Figure 10(A) Total GSH amount normalized to protein from kidney and (B) brain of wild-type (WT) and mutant (Mut) mice. Error bars represent SDs of at least 3 animals per group.(C) Thiosulfate levels in urine and (D) plasma normalized to protein amount. Error bars represent SDs of 9 animals per group (urine) and 6 animals per group (plasma).(E) H2S amount normalized to tissue from kidney and (F) brain. Error bars represent SDs of 7/8 animals per group.(G) Acylcarnitine C4, C5 and C6 levels from plasma, normalized to volume. Error bars represent SDs of 5 wild-type and 3 mutants.


Figure 1T2 - weighed brain MRIs of patient A ( D1 - 3 ) at 28 months of age and patient B at ( D4 - 6 ) at 16 months of age , showing global brain atrophy as well as periventricular and deep white matter hyperintensities .

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Figure 1T2-weighed brain MRIs of patient A (D1-3) at 28 months of age and patient B at (D4-6) at 16 months of age, showing global brain atrophy as well as periventricular and deep white matter hyperintensities.


Figure 3Quantifications of HOPS / CORVET subunits in fibroblast lysates ( top ) and representative immunoblots ( bottom ) , analyzed as in ( C ) . ( E ) VPS33A , ( F ) VPS11 , ( G ) VPS18 . Immunoblots ( left ) and quantifications normalized to control ( right ) of VPS16 , VPS33A and VPS11 in lysates from fibroblasts transduced with a lentivirus to express VPS16 or control ( empty vector ) .

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Figure 3Quantifications of HOPS/CORVET subunits in fibroblast lysates (top) and representative immunoblots (bottom), analyzed as in (C). (E) VPS33A, (F) VPS11, (G) VPS18.Immunoblots (left) and quantifications normalized to control (right) of VPS16, VPS33A and VPS11 in lysates from fibroblasts transduced with a lentivirus to express VPS16 or control (empty vector).


Figure 4Analysis of the early endosome marker EEA1 in fibroblasts transduced with control or VPS16 - expressing lentivirus by confocal microscopy . ( A ) Representative confocal micrographs . Scale bar , 20 μm . ( B ) Quantification of EEA1 - stained puncta number and intensities ( ca 80 cells analyzed for each of n = 3 biological replicates ) . Analysis of intracellular Transferrin trafficking . Fibroblasts fed with fluorescently labeled Transferrin were co - stained with LysoTracker ( G ) or an antibody against LAMP2 ( H ) . Representative images ( left ) of fibroblasts transduced with control virus , with magnified areas demonstrating co - localized puncta ( arrowheads ) . Scale bars 10 μm or 2 μm ( inserts ) . Quantifications ( right ) of the co - localization between Transferrin and LysoTracker or LAMP2 , respectively , expressed as Pearson correlation coefficients . Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles ( n = 10 optical sections from 3 independent experiments ) .

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Figure 4Analysis of the early endosome marker EEA1 in fibroblasts transduced with control or VPS16-expressing lentivirus by confocal microscopy. (A) Representative confocal micrographs. Scale bar, 20 μm. (B) Quantification of EEA1-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).Analysis of intracellular Transferrin trafficking. Fibroblasts fed with fluorescently labeled Transferrin were co-stained with LysoTracker (G) or an antibody against LAMP2 (H). Representative images (left) of fibroblasts transduced with control virus, with magnified areas demonstrating co-localized puncta (arrowheads). Scale bars 10 μm or 2 μm (inserts). Quantifications (right) of the co-localization between Transferrin and LysoTracker or LAMP2, respectively, expressed as Pearson correlation coefficients. Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles (n=10 optical sections from 3 independent experiments).


Figure 5Confocal micrographs of fibroblasts transduced with control or VPS16 - expressing lentivirus and labeled with LysoTracker . Scale bar , 20 μm . Quantification of LysoTracker - stained puncta number and intensities ( ca 80 cells analyzed for each of n = 3 biological replicates ) . Representative immunoblots ( bottom ) and summary quantifications ( top ) of the levels of LAMP1 ( C ) and LAMP2 ( D ) in fibroblasts , normalized to levels of actin ( ACTB ) and expressed as % of controls ( n = 3 biological replicates ) . Analysis of lysosomal Cathepsin D ( CtsD ) processing by immunoblotting . Immunoblot to detect CtsD isoforms ( left ) and quantification ( right ) of processing , calculated as the percentage of CtsD divided by the sum of all isoforms ( n = 3 biological replicates ) .

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Figure 5Confocal micrographs of fibroblasts transduced with control or VPS16-expressing lentivirus and labeled with LysoTracker. Scale bar, 20 μm. Quantification of LysoTracker-stained puncta number and intensities (ca 80 cells analyzed for each of n=3 biological replicates).Representative immunoblots (bottom) and summary quantifications (top) of the levels of LAMP1 (C) and LAMP2 (D) in fibroblasts, normalized to levels of actin (ACTB) and expressed as % of controls (n=3 biological replicates). Analysis of lysosomal Cathepsin D (CtsD) processing by immunoblotting. Immunoblot to detect CtsD isoforms (left) and quantification (right) of processing, calculated as the percentage of CtsD divided by the sum of all isoforms (n=3 biological replicates).


Figure 6Analysis of autophagosome numbers in fibroblasts transfected to express mRFP - GFP - LC3 . ( F ) Representative confocal micrographs ( optical sections ) of fibroblasts imaged live under basal or serum - starved conditions . Scale bar , 20 μm . Analysis of autophagosome numbers in fibroblasts transfected to express mRFP - GFP - LC3 . Quantification of autophagosomes , defined as green + red puncta ( G ) and autophago - lysosomes , calculated as the number of red ( but not green ) puncta divided by the total number of puncta . Colored horizontal bars denote median values and whiskers 5 and 95 percentiles ( n = 50 - 113 cells from a total of 3 independent experiments ; for exact values see Appendix Table S1 ) .

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Figure 6Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. (F) Representative confocal micrographs (optical sections) of fibroblasts imaged live under basal or serum-starved conditions. Scale bar, 20 μm.Analysis of autophagosome numbers in fibroblasts transfected to express mRFP-GFP-LC3. Quantification of autophagosomes, defined as green + red puncta (G) and autophago-lysosomes, calculated as the number of red (but not green) puncta divided by the total number of puncta. Colored horizontal bars denote median values and whiskers 5 and 95 percentiles (n=50-113 cells from a total of 3 independent experiments; for exact values see Appendix Table S1).


Figure 7Myelination , as reported using the Tg ( mbp : gfp - caax ) transgenic , is significantly reduced in vps16 crispants . The yellow box indicates the region assessed in D . Scale bar 100 μm . Bar graphs represent data as mean ± SEM . Statistical comparisons by Mann - Whitney U tests ( n = 6 ) . * * p < 0 . 01 .

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Figure 7Myelination, as reported using the Tg(mbp:gfp-caax) transgenic, is significantly reduced in vps16 crispants. The yellow box indicates the region assessed in D. Scale bar 100 μm. Bar graphs represent data as mean ±SEM. Statistical comparisons by Mann-Whitney U tests (n=6). ** p < 0.01.


Figure 8LysoTracker signal colocalizes with the astroglial reporter slc1a2b : citrine and does not appear to overlap with the pan - neuronal reporter elavl3 : GCaMP5G .

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Figure 8LysoTracker signal colocalizes with the astroglial reporter slc1a2b:citrine and does not appear to overlap with the pan-neuronal reporter elavl3:GCaMP5G.


Figure 1C - F . Immunofluorescence images of esg - GFP and Delta ( Dl ) staining with the midgut section from the R4 region of 14 - day Drosophila ( C ) , 26 - day Drosophila ( D ) , 40 - day Drosophila ( E ) , and 40 - day Drosophila in response to 0 . 5 mM ALA administration , which started at day 26 after eclosion ( F ) . esg - GFP ( green ) identifies ISCs and their differentiating cells . Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . J . Western blotting results of midguts indicate a decrease of LAS in the midguts of aged Drosophila . Loading controls , β - tubulin . K . Quantification of LAS band intensity as seen in experiments ( J ) . Error bars indicate the SD of three independent experiments . L Data information : Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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Figure 1C-F. Immunofluorescence images of esg-GFP and Delta (Dl) staining with the midgut section from the R4 region of 14-day Drosophila (C), 26-day Drosophila (D), 40-day Drosophila (E), and 40-day Drosophila in response to 0.5 mM ALA administration, which started at day 26 after eclosion (F). esg-GFP (green) identifies ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue.J. Western blotting results of midguts indicate a decrease of LAS in the midguts of aged Drosophila. Loading controls, β-tubulin. K. Quantification of LAS band intensity as seen in experiments (J). Error bars indicate the SD of three independent experiments. L Data information: Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


Figure 2C . Quantification of the content of ALA in midguts of flies with indicated genotypes using LC - ESI - MS / MS . Error bars indicate the SD of three independent experiments . Data information : Error bars represent SDs . Student ' s t - tests , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 . D - F . Immunofluorescence images of the midgut section from the R4 region in Drosophila carrying esgts - GAL4 - driven UAS - lacZ ( D , control ) , Las RNAi ( E ) , or Las RNAi in response to ALA administration ( F ) . esg - GFP ( green ) indicates ISCs and their differentiating cells . Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μmI - J . Immunofluorescence images of control ( FRT40A , I ) and Las RNAi ( J ) MARCM clones ( green , outlined by white dotted lines ) 10 days after clone induction ( ACI ) . Pdm1 staining ( red ) was used to visualize ECs . K . Quantification of the ratio of Pdm1 + cells per clone of MARCM clones with indicated genotypes . n is indicated . Each dot corresponds to one clone . L . Quantification of MARCM clone size of experiments in ( I - J ) . n is indicated . Each dot corresponds to one clone . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μm ( D Error bars represent SDs . Student ' s t - tests , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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Figure 2C. Quantification of the content of ALA in midguts of flies with indicated genotypes using LC-ESI-MS/MS. Error bars indicate the SD of three independent experiments. Data information: Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05.D-F. Immunofluorescence images of the midgut section from the R4 region in Drosophila carrying esgts-GAL4-driven UAS-lacZ (D, control), Las RNAi (E), or Las RNAi in response to ALA administration (F). esg-GFP (green) indicates ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μmI-J. Immunofluorescence images of control (FRT40A, I) and Las RNAi (J) MARCM clones (green, outlined by white dotted lines) 10 days after clone induction (ACI). Pdm1 staining (red) was used to visualize ECs. K. Quantification of the ratio of Pdm1+ cells per clone of MARCM clones with indicated genotypes. n is indicated. Each dot corresponds to one clone. L. Quantification of MARCM clone size of experiments in (I-J). n is indicated. Each dot corresponds to one clone. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm (D Error bars represent SDs. Student's t-tests, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


Figure 3A . Representative images and quantification of Drosophila midguts treated with the pH indicator Bromophenol Blue . Moreover , quantification of the three categories of Drosophila of indicated conditions . The three categories can be divided into : homeostasis , a well - defined acidic ( yellow colored ) copper cell region ( CCR ) is flanked by basic ( blue colored ) anterior midgut ( AM ) and posterior midgut ( PM ) . " Perturbed A " , the acidic region is lost and the whole gut is basic ; " Perturbed B " , the strongly acidic region is lost , and the remainder of the gut also becomes less basic . The numbers of quantified guts from left to right are 90 , 90 , and 90 . Error bars show the SD of three independent experiments . C . Excretion of Drosophila treated with Bromophenol Blue . Images of deposits and quantification of deposit numbers are shown . Excretions are quantified in 30 fields for each group of 12 Drosophila . Tests were repeated as three independent experiments . Data information : The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 . G . Survival ( percentage ) of female W1118 Drosophila with and without supplementation of ALA as indicated . The numbers of quantified flies : 100 ( W1118 + 0 . 5 mM ALA ) and 100 ( W1118 + 0 mM ALA ) . Three independent experiments were conducted . Data information : Error bars represent SDs . p - values for lifespan curves were calculated by the log - rank test . The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 . I . Survival ( percentage ) of female Canton - S Drosophila with and without supplementation of ALA as indicated . The numbers of quantified flies : 100 ( Canton - S + 0 mM ALA ) , 100 ( Canton - S + 0 . 5 mM ALA ) . Three independent experiments were conducted . J . Survival ( percentage ) of female Canton - S Drosophila with and without supplementation of ALA at 26 - day as indicated . The numbers of quantified flies : 100 ( Canton - S + 0 mM ALA ) and 100 ( Canton - S + 0 . 5 mM ALA ) . Three independent experiments were conducted . Data information : Error bars represent SDs . p - values for lifespan curves ( were calculated by the log - rank test . The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative images and quantification of Drosophila midguts treated with the pH indicator Bromophenol Blue. Moreover, quantification of the three categories of Drosophila of indicated conditions. The three categories can be divided into: homeostasis, a well-defined acidic (yellow colored) copper cell region (CCR) is flanked by basic (blue colored) anterior midgut (AM) and posterior midgut (PM). "Perturbed A", the acidic region is lost and the whole gut is basic; "Perturbed B", the strongly acidic region is lost, and the remainder of the gut also becomes less basic. The numbers of quantified guts from left to right are 90, 90, and 90. Error bars show the SD of three independent experiments.C. Excretion of Drosophila treated with Bromophenol Blue. Images of deposits and quantification of deposit numbers are shown. Excretions are quantified in 30 fields for each group of 12 Drosophila. Tests were repeated as three independent experiments. Data information: The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.G. Survival (percentage) of female W1118 Drosophila with and without supplementation of ALA as indicated. The numbers of quantified flies: 100 (W1118 + 0.5 mM ALA) and 100 (W1118 + 0 mM ALA). Three independent experiments were conducted. Data information: Error bars represent SDs. p-values for lifespan curves were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.I. Survival (percentage) of female Canton-S Drosophila with and without supplementation of ALA as indicated. The numbers of quantified flies: 100 (Canton-S + 0 mM ALA), 100 (Canton-S + 0.5 mM ALA). Three independent experiments were conducted. J. Survival (percentage) of female Canton-S Drosophila with and without supplementation of ALA at 26-day as indicated. The numbers of quantified flies: 100 (Canton-S + 0 mM ALA) and 100 (Canton-S + 0.5 mM ALA). Three independent experiments were conducted. Data information: Error bars represent SDs. p-values for lifespan curves ( were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


Figure 4A - F . Immunofluorescence images of midgut sections from the R4 region in Drosophila carrying esgts - GAL4 - driven expression of CAT cDNA ( A ) , CAT cDNA with AD4 administration ( B ) , CAT cDNA with ALA administration ( C ) , Keap1 RNAi ( D ) , Keap1 RNAi with AD4 administration ( E ) , or Keap1 RNAi with ALA administration ( F ) . esg - GFP ( green ) represents ISCs and their differentiating cells . Dl staining ( red ) was used to visualize ISCs . G . Quantification of the number of esg - GFP + cells , Dl + cells of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA , CAT cDNA , CAT cDNA with AD4 administration , CAT cDNA with ALA administration , Keap1 RNAi , Keap1 RNAi with AD4 administration , or Keap1 RNAi with ALA administration . n is indicated . The numbers of quantified guts from left to right are 18 , 19 , 20 , 18 , 18 , 19 , 19 , 18 , 19 , 20 , 18 , 18 , 19 , and 19 . H . Quantification of the number of pH3 + cells and luciferase activity of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA , CAT cDNA , CAT cDNA with AD4 administration , CAT cDNA with ALA administration , Keap1 RNAi , Keap1 RNAi with AD4 administration , or Keap1 RNAi with ALA administration . The numbers of quantified guts from left to right are 18 , 19 , 20 , 18 , 18 , 19 , and 19 . I - Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μm Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 . I - L . Representative images of the midgut section from the R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( I ) , Las RNAi ( J ) , Las RNAi and CAT cDNA ( K ) , or Las RNAi and Keap1 RNAi ( L ) . esg - GFP ( green ) indicates ISCs and their differentiating cells . Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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 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 4A-F. Immunofluorescence images of midgut sections from the R4 region in Drosophila carrying esgts-GAL4-driven expression of CAT cDNA (A), CAT cDNA with AD4 administration (B), CAT cDNA with ALA administration (C), Keap1 RNAi (D), Keap1 RNAi with AD4 administration (E), or Keap1 RNAi with ALA administration (F). esg-GFP (green) represents ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. G. Quantification of the number of esg-GFP+ cells, Dl+ cells of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA, CAT cDNA, CAT cDNA with AD4 administration, CAT cDNA with ALA administration, Keap1 RNAi, Keap1 RNAi with AD4 administration, or Keap1 RNAi with ALA administration. n is indicated. The numbers of quantified guts from left to right are 18, 19, 20, 18, 18, 19, 19, 18, 19, 20, 18, 18, 19, and 19. H. Quantification of the number of pH3+ cells and luciferase activity of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA, CAT cDNA, CAT cDNA with AD4 administration, CAT cDNA with ALA administration, Keap1 RNAi, Keap1 RNAi with AD4 administration, or Keap1 RNAi with ALA administration. The numbers of quantified guts from left to right are 18, 19, 20, 18, 18, 19, and 19. I- Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.I-L. Representative images of the midgut section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (I), Las RNAi (J), Las RNAi and CAT cDNA (K), or Las RNAi and Keap1 RNAi (L). esg-GFP (green) indicates ISCs and their differentiating cells. Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


Figure 5A . Volcano plots of differentially expressed genes in pair - wise comparison of 40 - day Drosophila treated without ALA administration to 40 - day Drosophila treated with 0 . 5 mM ALA administration . Blue symbols indicate significantly downregulated mass bins ( Log2 FC < - 0 . 6 and p < 0 . 05 ) , red symbols indicate significantly upregulated mass bins ( Log2 FC > 0 . 6 and p < 0 . 05 ) , and gray symbols indicate mass bins that were not significantly changed .

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

Figure 5A. Volcano plots of differentially expressed genes in pair-wise comparison of 40-day Drosophila treated without ALA administration to 40-day Drosophila treated with 0.5 mM ALA administration. Blue symbols indicate significantly downregulated mass bins (Log2 FC < -0.6 and p < 0.05), red symbols indicate significantly upregulated mass bins (Log2 FC > 0.6 and p < 0.05), and gray symbols indicate mass bins that were not significantly changed.


Figure 6A - C . Immunofluorescence images of esg - GFP and Lysotracker staining with the midgut section from the R4 region in 14 - day WT flies ( A ) , 40 - day WT flies ( B ) , 40 - day WT flies with ALA administration ( C ) . esg - GFP ( green ; outlined by dotted lines ) , lysotracker ( red ) . esg - GFP + cells are outlined by red dotted lines in the images of lysotracker staining channel . D . Quantification of the dot number of Lysotracker in esg positive cells from experiments ( A - C ) . n is indicated . The numbers of quantified guts from left to right are 9 , 11 , and 8 . E . Quantification of the dot size of Lysotracker in esg positive cells from experiments ( A - C ) . n is as indicated . F - Data information : DAPI stained nuclei are shown in blue . Scale bars represent 5 μm Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 . F - H . Expression of esg - GAL4 - driven UAS - GFP - mCherry - Atg8a in 14 - day Drosophila ( F ) , 40 - day Drosophila ( G ) , 40 - day Drosophila with ALA administration started at the middle age ( 26 days ) ( H ) . GFP ( green ) and mCherry ( red ) . The red arrows indicate the autophagosomes . I . Quantification of the dot number of mCherry in esg positive cells from experiments ( F - H ) . n is indicated . The numbers of quantified guts from left to right are 11 , 10 , and 12 . J . Quantification of the dot size of mCherry in esg positive cells from experiments ( F - H ) . n is as indicated . K - Data information : DAPI stained nuclei are shown in blue . Scale bars represent 2 μm Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 . K - L . Immunofluorescence images of esg - GFP and Lysotracker staining with the midgut section from the R4 region in 14 - day flies carrying esgts - GAL4 - driven UAS - lacZ ( K ) , and 14 - day flies carrying esgts - GAL4 - driven Las RNAi ( L ) . esg - GFP ( green ; outlined by dotted lines ) , lysotracker ( red ) . esg - GFP + cells are outlined by dotted lines . M . Quantification of the dot number of Lysotracker in esg positive cells from experiments ( K - L ) . n is indicated . The numbers of quantified guts from left to right are 10 , and 9 . N . Quantification of the dot size of Lysotracker in esg positive cells from experiments ( K - L ) . n is as indicated . O - Data information : DAPI stained nuclei are shown in blue . Scale bars represent 5 μm Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 . O - P . Expression of esg - GAL4 - driven UAS - GFP - mCherry - Atg8a in 14 - day Drosophila carrying esgts - GAL4 - driven UAS - lacZ ( flies were cultured at 18 ° C and transferred to 29 ° C after flies eclosion ) ( O ) , and 14 - day Drosophila with Las depleted in ISCs and EBs ( flies were cultured at 18 ° C and transferred to 29 ° C after flies eclosion ) ( P ) . GFP ( green ) and mCherry ( red ) . The red arrows indicate the autophagosomes . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 2 μm Error bars represent SDs . Student ' s t - tests , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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 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 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 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

Figure 6A-C. Immunofluorescence images of esg-GFP and Lysotracker staining with the midgut section from the R4 region in 14-day WT flies (A), 40-day WT flies (B), 40-day WT flies with ALA administration (C). esg-GFP (green; outlined by dotted lines), lysotracker (red). esg-GFP+ cells are outlined by red dotted lines in the images of lysotracker staining channel. D. Quantification of the dot number of Lysotracker in esg positive cells from experiments (A-C). n is indicated. The numbers of quantified guts from left to right are 9, 11, and 8. E. Quantification of the dot size of Lysotracker in esg positive cells from experiments (A-C). n is as indicated. F- Data information: DAPI stained nuclei are shown in blue. Scale bars represent 5 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.F-H. Expression of esg-GAL4-driven UAS-GFP-mCherry-Atg8a in 14-day Drosophila (F), 40-day Drosophila (G), 40-day Drosophila with ALA administration started at the middle age (26 days) (H). GFP (green) and mCherry (red). The red arrows indicate the autophagosomes. I. Quantification of the dot number of mCherry in esg positive cells from experiments (F-H). n is indicated. The numbers of quantified guts from left to right are 11, 10, and 12. J. Quantification of the dot size of mCherry in esg positive cells from experiments (F-H). n is as indicated. K- Data information: DAPI stained nuclei are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.K-L. Immunofluorescence images of esg-GFP and Lysotracker staining with the midgut section from the R4 region in 14-day flies carrying esgts-GAL4-driven UAS-lacZ (K), and 14-day flies carrying esgts-GAL4-driven Las RNAi (L). esg-GFP (green; outlined by dotted lines), lysotracker (red). esg-GFP+ cells are outlined by dotted lines. M. Quantification of the dot number of Lysotracker in esg positive cells from experiments (K-L). n is indicated. The numbers of quantified guts from left to right are 10, and 9. N. Quantification of the dot size of Lysotracker in esg positive cells from experiments (K-L). n is as indicated. O- Data information: DAPI stained nuclei are shown in blue. Scale bars represent 5 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.O-P. Expression of esg-GAL4-driven UAS-GFP-mCherry-Atg8a in 14-day Drosophila carrying esgts-GAL4-driven UAS-lacZ (flies were cultured at 18 °C and transferred to 29 °C after flies eclosion) (O), and 14-day Drosophila with Las depleted in ISCs and EBs (flies were cultured at 18 °C and transferred to 29 °C after flies eclosion) (P). GFP (green) and mCherry (red). The red arrows indicate the autophagosomes. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


Figure 7A - C . Immunofluorescence images of midgut section from the R4 region in 40 - day flies ( A ) , 40 - day flies with spermidine administration started at 26th day after fly eclosion ( B ) , 40 - day flies with spermidine and ALA administration started at 26th day after fly eclosion ( C ) . esg - GFP ( green ) indicates ISCs and their differentiating cells . Dl ( red ) staining was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μmF - I . Representative images of the midgut section from the R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( F , control ) , Las RNAi ( G ) , Las RNAi and Atg5 cDNA ( H ) , or Las RNAi with spermidine administration ( I ) . GFP ( green ) and Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μmN . Survival ( percentage ) of female Drosophila with indicated genotypes . The numbers of quantified Drosophila : 100 ( UAS - lacZ ) , 100 ( Las RNAi ) , 100 ( Las RNAi + UAS - Atg5 ) , and 100 ( Las RNAi + UAS - Atg8a ) . Three independent experiments were conducted . Data information : Error bars represent SDs . p - values for lifespan curves ( N ) were calculated by the log - rank test . The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 .

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 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 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

Figure 7A-C. Immunofluorescence images of midgut section from the R4 region in 40-day flies (A), 40-day flies with spermidine administration started at 26th day after fly eclosion (B), 40-day flies with spermidine and ALA administration started at 26th day after fly eclosion (C). esg-GFP (green) indicates ISCs and their differentiating cells. Dl (red) staining was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μmF-I. Representative images of the midgut section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (F, control), Las RNAi (G), Las RNAi and Atg5 cDNA (H), or Las RNAi with spermidine administration (I). GFP (green) and Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μmN. Survival (percentage) of female Drosophila with indicated genotypes. The numbers of quantified Drosophila: 100 (UAS-lacZ), 100 (Las RNAi), 100 (Las RNAi + UAS-Atg5), and 100 (Las RNAi + UAS-Atg8a). Three independent experiments were conducted. Data information: Error bars represent SDs. p-values for lifespan curves (N) were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.


Figure 8A - E . Expressions of Rab5 - GFP reporter in ISCs of 14 - day WT Drosophila ( A ) , 40 - day WT Drosophila ( B ) , 40 - day Drosophila with ALA administration ( C ) , 14 - day Drosophila carrying esgts - GAL4 > UAS - lacZ ( D ) , and 14 - day Drosophila carrying esgts - GAL4 > Las RNAi ( E ) . GFP ( green ) . F . Quantification of fluorescence intensity of RAB5 - GFP in experiments ( A - E ) . Each dot corresponds to one cell . n is indicated . The numbers of quantified guts from left to right are 13 , 11 , 12 , 10 and 13 . G Data information : DAPI stained nuclei are shown in blue . Scale bars represent 2 μm Error bars represent SDs . Student ' s t - tests , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 . G - K . Expressions of Rab7 - GFP reporter in ISCs of 14 - day WT Drosophila ( G ) , 40 - day WT Drosophila ( H ) , 40 - day Drosophila with ALA administration ( I ) , 14 - day Drosophila carrying esgts - GAL4 > UAS - lacZ ( J ) , and 14 - day Drosophila carrying esgts - GAL4 > Las RNAi ( K ) . GFP ( green ) . L . Quantification of fluorescence intensity of RAB7 - GFP in experiments ( G - K ) . Each dot corresponds to one cell . n is indicated . The numbers of quantified guts from left to right are 11 , 10 , 11 , 13 and 12 . M Data information : DAPI stained nuclei are shown in blue . Scale bars represent 2 μm Error bars represent SDs . Student ' s t - tests , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 . M - Q . Immunofluorescence images of midgut section from the R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( M , control ) , constitutively active form of RAB5 ( N ) , constitutively active form of RAB5 with ALA administration ( O ) , constitutively active form of RAB7 ( P ) , or constitutively active form of RAB7 with ALA administration ( Q ) . GFP ( green ) and Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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 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 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

Figure 8A-E. Expressions of Rab5-GFP reporter in ISCs of 14-day WT Drosophila (A), 40-day WT Drosophila (B), 40-day Drosophila with ALA administration (C), 14-day Drosophila carrying esgts-GAL4>UAS-lacZ (D), and 14-day Drosophila carrying esgts-GAL4>Las RNAi (E). GFP (green). F. Quantification of fluorescence intensity of RAB5-GFP in experiments (A-E). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 13, 11, 12, 10 and 13. G Data information: DAPI stained nuclei are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.G-K. Expressions of Rab7-GFP reporter in ISCs of 14-day WT Drosophila (G), 40-day WT Drosophila (H), 40-day Drosophila with ALA administration (I), 14-day Drosophila carrying esgts-GAL4>UAS-lacZ (J), and 14-day Drosophila carrying esgts-GAL4>Las RNAi (K). GFP (green). L. Quantification of fluorescence intensity of RAB7-GFP in experiments (G-K). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 11, 10, 11, 13 and 12. M Data information: DAPI stained nuclei are shown in blue. Scale bars represent 2 μm Error bars represent SDs. Student's t-tests, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.M-Q. Immunofluorescence images of midgut section from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (M, control), constitutively active form of RAB5 (N), constitutively active form of RAB5 with ALA administration (O), constitutively active form of RAB7 (P), or constitutively active form of RAB7 with ALA administration (Q). GFP (green) and Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


Figure 9A - D . Representative images of midgut sections from the R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( A , control ) , Las RNAi ( B ) , Las RNAi and RAB5 - CA ( C ) , or Las RNAi and RAB7 - CA ( D ) . GFP ( green ) , Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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

Figure 9A-D. Representative images of midgut sections from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (A, control), Las RNAi (B), Las RNAi and RAB5-CA (C), or Las RNAi and RAB7-CA (D). GFP (green), Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


Figure 10C . Survival ( percentage ) of female Drosophila of indicated genotypes . The numbers of quantified Drosophila : 100 ( UAS - lacZ ) , 100 ( Las RNAi ) , 100 ( Las RNAi + UAS - Rab5 - CA ) and 100 ( Las RNAi + UAS - Rab7 - CA ) . Three independent experiments were conducted Data information : Error bars represent SDs . p - values for lifespan curves ( C ) were calculated by the log - rank test . The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 . F - I . Immunofluorescence images of dpERK staining of midgut sections of the R4 region of 40 - day Drosophila ( F ) , 40 - day Drosophila with ALA administration ( G ) , 14 - day Drosophila carrying esgts - GAL4 - driven UAS - lacZ ( H ) , and Drosophila carrying esgts - GAL4 - driven Las RNAi ( I ) . dpERK ( red ) . J . Quantification of the fluorescence intensity of dpERK in experiments ( F - I ) . Each dot corresponds to one cell . n is indicated . The numbers of quantified guts from left to right are 11 , 12 , 12 , and 13 . K Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10 μm Error bars represent SDs . The statistical tests used in other panels were student ' s t - tests . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , and non - significant ( NS ) represents p > 0 . 05 . K - M . Representative images of midgut sections from the R4 region of Drosophila carrying esgts - GAL4 - driven expression of lacZ cDNA ( K , control ) , Las RNAi ( L ) , or Las RNAi and dominant negative form of EGFR ( EGFR - DN , M ) . GFP ( green ) and Dl staining ( red ) was used to visualize ISCs . Data information : DAPI stained nuclei are shown in blue . Scale bars represent 10

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 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 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

Figure 10C. Survival (percentage) of female Drosophila of indicated genotypes. The numbers of quantified Drosophila: 100 (UAS-lacZ), 100 (Las RNAi), 100 (Las RNAi + UAS-Rab5-CA) and 100 (Las RNAi + UAS-Rab7-CA). Three independent experiments were conducted Data information: Error bars represent SDs. p-values for lifespan curves (C) were calculated by the log-rank test. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.F-I. Immunofluorescence images of dpERK staining of midgut sections of the R4 region of 40-day Drosophila (F), 40-day Drosophila with ALA administration (G), 14-day Drosophila carrying esgts-GAL4-driven UAS-lacZ (H), and Drosophila carrying esgts-GAL4-driven Las RNAi (I). dpERK (red). J. Quantification of the fluorescence intensity of dpERK in experiments (F-I). Each dot corresponds to one cell. n is indicated. The numbers of quantified guts from left to right are 11, 12, 12, and 13. K Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm Error bars represent SDs. The statistical tests used in other panels were student's t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and non-significant (NS) represents p > 0.05.K-M. Representative images of midgut sections from the R4 region of Drosophila carrying esgts-GAL4-driven expression of lacZ cDNA (K, control), Las RNAi (L), or Las RNAi and dominant negative form of EGFR (EGFR-DN, M). GFP (green) and Dl staining (red) was used to visualize ISCs. Data information: DAPI stained nuclei are shown in blue. Scale bars represent 10 μm


Figure 1 ( A ) ATG - 5 staining in non - cancerous gastric tissues scored 285 ( × 200 ) ; ( B ) ATG - 5 staining in GC tumor tissues scored 50 ( × 400 ) ; ( C ) ATG - 5 staining in GC tumor tissues scored 270 ( × 400 ) ; ( D ) ATG - 5 staining in GC tumor tissues scored 400 ( × 200 ) .

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Figure 1  (A) ATG-5 staining in non-cancerous gastric tissues scored 285(×200); (B) ATG-5 staining in GC tumor tissues scored 50(×400); (C) ATG-5 staining in GC tumor tissues scored 270(×400); (D) ATG-5 staining in GC tumor tissues scored 400(×200).


Figure 2 ( A and B ) Patients were divided into four following subgroups based on ATG - 5 immunostaining : scored 0 - 99 ( curve a ) , scored 100 - 199 ( curve b ) , scored 200 - 299 ( curve c ) and scored 300 - 400 ( curve d ) . The difference among different subgroups was statistically significant as evaluated by overall Log - rank comparisons ( OS : P < 0 . 001 , DFS : P = 0 . 003 ) . Pairwise Log - rank comparisons showed that the subgroup D exhibited the poorest survival rates as compared to other subgroups ( OS : P < 0 . 05 , DFS : P < 0 . 01 ) . ( C and D ) Patients were divided into four following subgroups based on MRP - 1 immunostaining : scored 0 - 99 ( curve a ) , scored 100 - 199 ( curve b ) , scored 200 - 299 ( curve c ) and scored 300 - 400 ( curve d ) . The difference among various subgroups was statistically significant by overall Log - rank comparisons ( OS : P = 0 . 001 , DFS : P = 0 . 018 ) . Pairwise Log - rank comparisons showed that the subgroup A had the most favorable prognosis among the four subgroups ( OS : P < 0 . 05 , DFS : P < 0 . 001 ) . ( E and F ) Patients were divided into four subgroups according to different TNM stages . The difference among different subgroups was statistically significant by overall Log - rank comparisons ( OS : P < 0 . 001 , DFS : P < 0 . 001 ) . Pairwise Log - rank comparisons showed that subgroups III or IV exhibited poorer survival rates than that of subgroups IB and II ( OS : P < 0 . 01 , DFS : P < 0 . 001 ) .

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 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 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

Figure 2(A and B) Patients were divided into four following subgroups based on ATG-5 immunostaining: scored 0-99 (curve a), scored 100-199 (curve b), scored 200-299 (curve c) and scored 300-400 (curve d). The difference among different subgroups was statistically significant as evaluated by overall Log-rank comparisons (OS: P<0.001, DFS: P = 0.003). Pairwise Log-rank comparisons showed that the subgroup D exhibited the poorest survival rates as compared to other subgroups (OS: P<0.05, DFS: P<0.01).(C and D) Patients were divided into four following subgroups based on MRP-1 immunostaining: scored 0-99 (curve a), scored 100-199 (curve b), scored 200-299 (curve c) and scored 300-400 (curve d). The difference among various subgroups was statistically significant by overall Log-rank comparisons (OS: P = 0.001, DFS: P = 0.018). Pairwise Log-rank comparisons showed that the subgroup A had the most favorable prognosis among the four subgroups (OS: P<0.05, DFS: P<0.001).(E and F) Patients were divided into four subgroups according to different TNM stages. The difference among different subgroups was statistically significant by overall Log-rank comparisons (OS: P<0.001, DFS: P<0.001). Pairwise Log-rank comparisons showed that subgroups III or IV exhibited poorer survival rates than that of subgroups IB and II (OS: P<0.01, DFS: P<0.001).


Figure 3 ( A ) The level of ATG5 was detected in cell lines using western blot analysis . β - actin was used as internal controls . ( B ) The IC 25 , IC50 and IC75 of SGC - 7901 and SGC - 7901 / DDP cells were tested using the MTT assays after DPP treatment .

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 O O O O O O O

Figure 3(A) The level of ATG5 was detected in cell lines using western blot analysis. β -actin was used as internal controls.(B) The IC 25, IC50 and IC75 of SGC-7901 and SGC-7901/DDP cells were tested using the MTT assays after DPP treatment.


Figure 4 ( A ) The mRNA level of ATG - 5 was detected by real time PCR after treatment with siRNAs . GAPDH was used as internal controls . ( B ) The protein level of ATG - 5 was detected by western blot after treatment with siRNAs . β - actin was used as internal controls . ( C ) The proliferation ability was tested using MTT assay 48 hours or 72 hours after different treatment .

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Figure 4(A) The mRNA level of ATG-5 was detected by real time PCR after treatment with siRNAs. GAPDH was used as internal controls.(B) The protein level of ATG-5 was detected by western blot after treatment with siRNAs. β -actin was used as internal controls.(C) The proliferation ability was tested using MTT assay 48 hours or 72 hours after different treatment.


Figure 5 ( A ) The autophagy was detected by immunofluorescence assay of LC3B in the cells 48 hours after different treatment . ( B ) The protein levels of LC3A and LC3B were tested by western blot . β - actin was used as internal controls .

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Figure 5(A) The autophagy was detected by immunofluorescence assay of LC3B in the cells 48 hours after different treatment.(B) The protein levels of LC3A and LC3B were tested by western blot. β-actin was used as internal controls.


Figure 1 ( c ) Intravascular endothelial cell interactions and transmigration of neutrophils ( Ly - 6G + CX3CR - 1 - ; red ) and cMOs ( Ly - 6G - CX3CR - 1low ; green ) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA , PAI - 1 , or uPA - PAI - 1 in postcapillary venules of the cremaster of CX3CR - 1GFP / + mice by multi - channel in vivo microscopy . Representative still images ( scale bar : 50 : µm ) and quantitative data are shown ( mean ± SEM for n = 4 mice per group ; # p < 0 . 05 vs . saline ; * p < 0 . 05 vs . uPA ; § p < 0 . 05 vs . PAI - 1 ; One - way ANOVA ) .

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

Figure 1(c) Intravascular endothelial cell interactions and transmigration of neutrophils (Ly-6G+ CX3CR-1-; red) and cMOs (Ly-6G- CX3CR-1low; green) to the perivascular tissue as assessed 6 h after intrascrotal stimulation with recombinant murine uPA, PAI-1, or uPA-PAI-1 in postcapillary venules of the cremaster of CX3CR-1GFP/+ mice by multi-channel in vivo microscopy. Representative still images (scale bar: 50: µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; *p<0.05 vs. uPA; §p<0.05 vs. PAI-1; One-way ANOVA).


Figure 2 ( b ) Expression of PECAM - 1 / CD31 ( red ) and ICAM - 1 / CD54 or VCAM - 1 / CD106 ( green ) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA - PAI - 1 or saline , representative images ( scale bar : 50 µm ) and quantitative data are shown ( mean ± SEM for n = 4 mice per group ; # p < 0 . 05 vs . saline ; t test ) . ( g ) Quantitative analysis of LFA - 1 / CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E - selectin / CD62E + ICAM - 1 / CD54 , E - selectin / CD62E + ICAM - 1 / CD54 + KC , or E - selectin / CD62E + ICAM - 1 / CD54 + KC + uPA - PAI - 1 as assessed by spinning disk confocal microscopy , representative images and quantitative data are shown ( scale bar : 10 μm ; mean ± SD ; n = 3 mice ; n = 10 - 13 flow chambers , n = 122 - 222 cells ) ; * p < 0 . 05 vs . E - selectin / CD62E + ICAM - 1 / CD54 ; One - way ANOVA ) .

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 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

Figure 2(b) Expression of PECAM-1/CD31 (red) and ICAM-1/CD54 or VCAM-1/CD106 (green) in the cremaster muscle of WT mice as assessed ex vivo by confocal laser scanning microscopy 6 h after intrascrotal injection of recombinant murine uPA-PAI-1 or saline, representative images (scale bar: 50 µm) and quantitative data are shown (mean±SEM for n=4 mice per group; #p<0.05 vs. saline; t test).(g) Quantitative analysis of LFA-1/CD11a clustering on the surface of mouse neutrophils under increasing flow conditions in vitro in the presence of either E-selectin/CD62E + ICAM-1/CD54, E-selectin/CD62E + ICAM-1/CD54 + KC, or E-selectin/CD62E + ICAM-1/CD54 + KC + uPA-PAI-1 as assessed by spinning disk confocal microscopy, representative images and quantitative data are shown (scale bar: 10 μm; mean±SD; n=3 mice; n=10-13 flow chambers, n =122-222 cells); *p<0.05 vs. E-selectin/CD62E + ICAM-1/CD54; One-way ANOVA).


Figure 3 ( b ) Correlation analyses of RNA expression levels of uPA ( PLAU gene ) , PAI - 1 ( SERPINE1 gene ) , and the neutrophil marker gene FPR1 , and overall survival of breast cancer patients ( stages 0 and 1 ) with high and low PLAU or SERPINE1 gene expression levels ( cut - off z ≥ 2 . 0 ) in the METABRIC breast cancer cohort . ( c ) Surface expression of MMP - 9 , VEGF , and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice ( saline ) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA - PAI - 1 ( uPA - PAI - 1 ) by multi - channel flow cytometry , quantitative data are shown ( mean ± SEM for n = 4 - 6 mice per group ; # p < 0 . 05 vs . saline ; t test ) . ( f ) Formation of NETs ( histone H3 + ; green ) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra - scrotal injection of uPA - PAI - 1 , TNF , or saline , PECAM - 1 / CD31 + postcapillary venules ( blue ) and Ly - 6G + neutrophils ( red ) are depicted . Representative images are shown ( scale bar : 50 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 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 O O O O O O O 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(b) Correlation analyses of RNA expression levels of uPA (PLAU gene), PAI-1 (SERPINE1 gene), and the neutrophil marker gene FPR1, and overall survival of breast cancer patients (stages 0 and 1) with high and low PLAU or SERPINE1 gene expression levels (cut-off z≥ 2.0) in the METABRIC breast cancer cohort.(c) Surface expression of MMP-9, VEGF, and NE as assessed on circulating neutrophils isolated from the peripheral blood of WT mice (saline) or from the peritoneal cavity of WT mice 6 h after intraperitoneal stimulation with uPA-PAI-1 (uPA-PAI-1) by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; #p<0.05 vs. saline; t test).(f) Formation of NETs (histone H3+; green) as assessed ex vivo by confocal microscopy in the cremaster muscle of WT mice 6 h after intra-scrotal injection of uPA-PAI-1, TNF, or saline, PECAM-1/CD31+ postcapillary venules (blue) and Ly-6G+ neutrophils (red) are depicted. Representative images are shown (scale bar: 50 µm).


Figure 4 ( a ) Effect of compound WX - 340 on binding of recombinant murine uPA to PAI - 1 protein as assessed by ELISA , quantitative data are shown ( mean ± SEM for n = 3 experiments per group ; # p < 0 . 05 vs . drug vehicle ; One - way ANOVA ) . ( d ) Relative development rates and tumor weight in animals treated with WX - 340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice ( mean ± SEM for n = 4 - 6 mice per group ; * p < 0 . 05 vs . drug vehicle ; One - way ANOVA ) . ( f ) Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX - 340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi - channel flow cytometry , quantitative data are shown ( mean ± SEM for n = 4 - 6 mice per group ; * p < 0 . 05 vs . drug vehicle ; 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 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 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

Figure 4(a) Effect of compound WX-340 on binding of recombinant murine uPA to PAI-1 protein as assessed by ELISA, quantitative data are shown (mean±SEM for n=3 experiments per group; #p<0.05 vs. drug vehicle; One-way ANOVA).(d) Relative development rates and tumor weight in animals treated with WX-340 a priori or therapeutically after 1 week after tumor cell injection on a daily basis as assessed in an orthotopic model of 4T1 breast cancer in WT mice (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; One-way ANOVA).(f) Numbers of neutrophils and 4T1 tumor cells in lungs and brain of WT mice receiving WX-340 or vehicle therapeutically after 1 week after tumor cell injection on a daily basis as assessed 14 days after intravenous injection of 4T1 tumor cells by multi-channel flow cytometry, quantitative data are shown (mean±SEM for n=4-6 mice per group; *p<0.05 vs. drug vehicle; t test).


Figure 1 ( A ) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene ( DVGlut - GAL4 > UAS - RFP ) , dNmnat ( DVGlut - Gal4 > UAS - dNmnat ) , or Wnd ( DVGlut - Gal4 > UAS - Wnd ) . ( B ) Quantification of mean ( ± SEM ) EPSP amplitudes , in which 75 consecutive evoked events were averaged per cell , and then cell amplitudes were averaged per genotype . [ UAS - RFP n = 13 cells , UAS - dNmnat n = 14 cells , UAS - Wnd n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 36 , F = 12 . 66 , p < 0 . 0001 . UAS - RFP vs UAS - dNmnat p < 0 . 0001 ( * * * * ) , UAS - RFP vs UAS - Wnd p = 0 . 0093 ( * * ) ] . ] . ( C ) Quantification of mean ( ± SEM ) mEPSP amplitudes , in which 75 consecutive spontaneous events were averaged per cell , and then cell amplitudes were averaged per genotype . [ UAS - RFP n = 15 cells , UAS - dNmnat n = 14 cells , UAS - Wnd n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 38 , F = 11 . 66 , p = 0 . 0001 . UAS - RFP vs UAS - dNmnat p = 0 . 994 ( NS ) , UAS - RFP vs UAS - Wnd p = 0 . 0003 ( * * * ) ] . ( D ) Quantification of quantal content , which was calculated individually per cell by dividing the mean EPSP amplitude by the mean mEPSP amplitude , and then averaged per genotype . [ UAS - RFP n = 15 cells , UAS - dNmnat n = 14 cells , UAS - Wnd n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 38 , F = 9 . 286 , p = 0 . 0006 . UAS - RFP vs UAS - dNmnat p = 0 . 0015 ( * * ) , UAS - RFP vs UAS - Wnd p = 0 . 0003 ( * * * ) ] . ] . ( E ) Representative images of the NMJ synaptic terminal at muscle 4 in third - instar larvae . The genotypes in this experiment were identical to those tested in the electrophysiology experiments in A - D . NMJs were stained for the presynaptic bouton marker DVGLUT ( green ) and nerve membrane marker HRP ( red ) . ( F ) Quantification of the mean ( ± SEM ) number of DVGlut + boutons per muscle 4 NMJ in each genotype . [ UAS - RFP n = 23 , UAS - dNmnat n = 21 , UAS - Wnd n = 20 NMJs . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 63 , F = 68 . 31 , p < 0 . 0001 . UAS - RFP vs UAS - dNmnat p = 0 . 9818 ( NS ) , UAS - RFP vs UAS - Wnd p < 0 . 0001 ( * * * * ) ] .

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 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 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 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 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

Figure 1(A) Representative EPSP and mEPSP electrophysiological traces from larval NMJs at Muscle 6 in which a motoneuron specific driver was used to overexpress either a control transgene (DVGlut-GAL4 > UAS-RFP), dNmnat (DVGlut-Gal4 > UAS-dNmnat), or Wnd (DVGlut-Gal4 > UAS-Wnd).(B) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 13 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=36, F=12.66, p<0.0001. UAS-RFP vs UAS-dNmnat p < 0.0001 (****), UAS-RFP vs UAS-Wnd p = 0.0093 (**)].]. (C) Quantification of mean (±SEM) mEPSP amplitudes, in which 75 consecutive spontaneous events were averaged per cell, and then cell amplitudes were averaged per genotype. [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F=11.66, p = 0.0001. UAS-RFP vs UAS-dNmnat p=0.994 (NS), UAS-RFP vs UAS-Wnd p = 0.0003 (***)].(D) Quantification of quantal content, which was calculated individually per cell by dividing the mean EPSP amplitude by the mean mEPSP amplitude, and then averaged per genotype. [UAS-RFP n = 15 cells, UAS-dNmnat n = 14 cells, UAS-Wnd n= 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F=9.286, p = 0.0006. UAS-RFP vs UAS-dNmnat p=0.0015 (**), UAS-RFP vs UAS-Wnd p = 0.0003 (***)].]. (E) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in the electrophysiology experiments in A-D. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red).(F) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [UAS-RFP n = 23, UAS-dNmnat n = 21, UAS-Wnd n= 20 NMJs. 1-way ANOVA w/Tukey's multiple comparisons, DF=63, F=68.31, p < 0.0001. UAS-RFP vs UAS-dNmnat p=0.9818 (NS), UAS-RFP vs UAS-Wnd p < 0.0001(****)].


Figure 2 ( A ) Representative EPSP and mEPSP physiological traces from larval NMJs at Muscle 6 . Recordings were taken from WT control larvae ( Elav - Gal4 > UAS - RFP ) , hiw mutants driving a control transgene ( hiwND8 ; Elav - Gal4 > UAS - RFP ) , and hiw mutants expressing RNAis to knockdown either dNmnat ( hiwND8 ; Elav - Gal4 > UAS - dNmnat - RNAi [ # 29402 ] ) or Wnd ( hiwND8 ; Elav - Gal4 > UAS - Wnd - RNAi [ # 25396 ] ) . ( B ) Quantification of mean ( ± SEM ) EPSP amplitudes , in which 75 consecutive evoked events were averaged per cell , and then cell amplitudes were averaged per genotype . [ WT n = 10 cells , hiw n = 10 cells , hiw > Wnd - RNAi n = 10 cells , hiw > dNmnat - RNAi n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 39 , F = 14 . 02 , p < 0 . 0001 . WT vs hiw p = 0 . 0012 ( * * ) , WT vs hiw > Wnd - RNAi p < 0 . 0001 ( * * * * ) , WT vs hiw > dNmnat - RNAi p = 0 . 9880 ( NS ) ] . ( C ) Quantification of quantal content ( ± SEM ) . [ WT n = 9 cells , hiw n = 9 cells , hiw > Wnd - RNAi n = 10 cells , hiw > dNmnat - RNAi n = 10 cells . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 38 , F = 2 . 508 , p < 0 . 0001 . WT vs hiw p = 0 . 0361 ( * ) , WT vs hiw > Wnd - RNAi p = 0 . 0001 ( * * * ) , WT vs hiw > dNmnat - RNAi p = 0 . 9889 ( NS ) ] . ( D ) Representative images of the NMJ synaptic terminal at muscle 4 in third - instar larvae . The genotypes in this experiment were identical to those tested in panels A - C . NMJs were stained for the presynaptic bouton marker DVGLUT ( green ) and nerve membrane marker HRP ( red ) . ( E ) Quantification of the mean ( ± SEM ) number of DVGlut + boutons per muscle 4 NMJ in each genotype . [ WT n = 23 , hiw n = 21 , hiw > Wnd - RNAi n = 16 , hiw > dNmnat - RNAi n = 19 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 78 , F = 169 . 1 , p < 0 . 0001 . WT vs hiw p < 0 . 0001 ( * * * * ) , WT vs hiw > Wnd - RNAi p = 0 . 9988 ( NS ) , WT vs hiw > dNmnat - RNAi p < 0 . 0001 ( * * * * ) , hiw vs hiw > dNmnat - RNAi p = 0 . 9695 ( NS ) ] .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative EPSP and mEPSP physiological traces from larval NMJs at Muscle 6. Recordings were taken from WT control larvae (Elav-Gal4>UAS-RFP), hiw mutants driving a control transgene (hiwND8;Elav-Gal4 > UAS-RFP), and hiw mutants expressing RNAis to knockdown either dNmnat (hiwND8;Elav-Gal4 > UAS-dNmnat-RNAi [#29402] ) or Wnd (hiwND8;Elav-Gal4 > UAS-Wnd-RNAi [#25396]).(B) Quantification of mean (±SEM) EPSP amplitudes, in which 75 consecutive evoked events were averaged per cell, and then cell amplitudes were averaged per genotype. [WT n = 10 cells, hiw n = 10 cells, hiw > Wnd-RNAi n= 10 cells, hiw > dNmnat-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=39, F=14.02, p<0.0001. WT vs hiw p = 0.0012 (**), WT vs hiw > Wnd-RNAi p < 0.0001 (****), WT vs hiw > dNmnat-RNAi p = 0.9880 (NS)].(C) Quantification of quantal content (±SEM). [WT n = 9 cells, hiw n = 9 cells, hiw > Wnd-RNAi n= 10 cells, hiw > dNmnat-RNAi n = 10 cells. 1-way ANOVA w/Tukey's multiple comparisons, DF=38, F= 2.508, p<0.0001. WT vs hiw p = 0.0361 (*), WT vs hiw > Wnd-RNAi p = 0.0001 (***), WT vs hiw > dNmnat-RNAi p = 0.9889 (NS)].(D) Representative images of the NMJ synaptic terminal at muscle 4 in third-instar larvae. The genotypes in this experiment were identical to those tested in panels A-C. NMJs were stained for the presynaptic bouton marker DVGLUT (green) and nerve membrane marker HRP (red).(E) Quantification of the mean (±SEM) number of DVGlut+ boutons per muscle 4 NMJ in each genotype. [WT n = 23, hiw n = 21 , hiw > Wnd-RNAi n= 16, hiw > dNmnat-RNAi n=19. 1-way ANOVA w/Tukey's multiple comparisons, DF=78, F=169.1, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > Wnd-RNAi p = 0.9988 (NS), WT vs hiw > dNmnat-RNAi p < 0.0001 (****), hiw vs hiw > dNmnat-RNAi p = 0.9695 (NS)].


Figure 3 ( A ) Representative images of immunostained larval NMJs and active zones from WT , hiw mutant , and hiw mutant driving an RNAi against dNmnat [ # 29402 ] . In the top 3 panels , greyscale images of entire terminal were acquired by staining for nerve marker HRP . Presynaptic Brp puncta ( green ) and postsynaptic dGluRIII clusters ( magenta ) were imaged to quantify N in each genotype . ( B ) Quantification of NMJ synaptic terminal surface area in each genotype . [ WT n = 14 , hiw n = 12 , hiw > dNmnat - RNAi n = 12 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 37 , F = 22 . 427 , p < 0 . 0001 . WT vs hiw p < 0 . 0001 ( * * * * ) , WT vs hiw > dNmnat - RNAi p < 0 . 0001 ( * * * * ) , hiw vs hiw > dNmnat - RNAi p = 0 . 2907 ( NS ) ] . ( C ) Quantification of BRP puncta per NMJ [ WT n = 14 , hiw n = 12 , hiw > dNmnat - RNAi n = 12 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 37 , F = 2 . 854 , p = 0 . 0711 . WT vs hiw p = 0 . 0573 ( NS ) , WT vs hiw > dNmnat - RNAi p = 0 . 4529 ( NS ) , hiw vs hiw > dNmnat - RNAi p = 0 . 5030 ( NS ) ] . ( D ) Quantification of percentage of Brp puncta that were apposed to a dGluRIII clusters at each NMJ [ WT n = 14 , hiw n = 12 , hiw > dNmnat - RNAi n = 12 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 37 , F = 2 . 093 , p = 0 . 4716 . WT vs hiw p = 0 . 2168 ( NS ) , WT vs hiw > dNmnat - RNAi p = 0 . 1882 ( NS ) , hiw vs hiw > dNmnat - RNAi p = 0 . 9966 ( NS ) ] . ( E ) Representative electrophysiological traces from a synaptic facilitation experiment in which trains of 5 pulses were used to evoke consecutive action potentials at 100 ms ISI . Genotypes for these experiments were : WT ( elav > UAS - RFP ) , hiw mutant ( hiw ; elav > UAS - RFP ) , or hiw depleted of dNmnat ( hiw ; elav > dNmnat - RNAi ) . ( F ) Quantification of the facilitation index , in which the amplitude of the 5th pulse is divided by the amplitude of the 1st pulse . [ 100 ms ISI : WT n = 11 , hiw n = 9 , hiw > dNmnat - RNAi n = 12 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 31 , F = 7 . 546 , p = 0 . 0023 . WT vs hiw p = 0 . 0020 ( * * ) , hiw vs hiw > dNmnat - RNAi p = 0 . 0229 ( * ) , WT vs hiw > dNmnat - RNAi p = 0 . 5141 ( NS ) . 50 ms ISI : WT n = 11 , hiw n = 9 , hiw > dNmnat - RNAi n = 12 . 1 - way ANOVA w / Tukey ' s multiple comparisons , DF = 31 , F = 5 . 587 , p = 0 . 0089 . WT vs hiw p = 0 . 0129 ( * ) , hiw vs hiw > dNmnat - RNAi p = 0 . 0217 ( * ) , WT vs hiw > dNmnat - RNAi p = 0 . 9546 ( NS ) . ]

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 O O O O O O O O O O O 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 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 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

Figure 3(A) Representative images of immunostained larval NMJs and active zones from WT, hiw mutant, and hiw mutant driving an RNAi against dNmnat [#29402]. In the top 3 panels, greyscale images of entire terminal were acquired by staining for nerve marker HRP. Presynaptic Brp puncta (green) and postsynaptic dGluRIII clusters (magenta) were imaged to quantify N in each genotype.(B) Quantification of NMJ synaptic terminal surface area in each genotype. [WT n = 14, hiw n = 12 , hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=22.427, p < 0.0001. WT vs hiw p < 0.0001 (****), WT vs hiw > dNmnat-RNAi p < 0.0001 (****), hiw vs hiw > dNmnat-RNAi p = 0.2907 (NS)].(C) Quantification of BRP puncta per NMJ [WT n = 14, hiw n = 12, hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=2.854, p = 0.0711. WT vs hiw p = 0.0573 (NS), WT vs hiw > dNmnat-RNAi p = 0.4529 (NS), hiw vs hiw > dNmnat-RNAi p = 0.5030 (NS)].(D) Quantification of percentage of Brp puncta that were apposed to a dGluRIII clusters at each NMJ [WT n = 14, hiw n = 12, hiw > dNmnat-RNAi n=12. 1-way ANOVA w/Tukey's multiple comparisons, DF=37, F=2.093, p = 0.4716. WT vs hiw p = 0.2168 (NS), WT vs hiw > dNmnat-RNAi p = 0.1882 (NS), hiw vs hiw > dNmnat-RNAi p = 0.9966 (NS)].(E) Representative electrophysiological traces from a synaptic facilitation experiment in which trains of 5 pulses were used to evoke consecutive action potentials at 100 ms ISI. Genotypes for these experiments were: WT (elav>UAS-RFP), hiw mutant (hiw; elav>UAS-RFP), or hiw depleted of dNmnat (hiw; elav>dNmnat-RNAi). (F) Quantification of the facilitation index, in which the amplitude of the 5th pulse is divided by the amplitude of the 1st pulse. [100 ms ISI: WT n=11, hiw n= 9, hiw>dNmnat-RNAi n= 12. 1-way ANOVA w/Tukey's multiple comparisons, DF = 31, F= 7.546, p =0.0023. WT vs hiw p =0.0020 (**), hiw vs hiw> dNmnat-RNAi p= 0.0229 (*), WT vs hiw> dNmnat-RNAi p= 0.5141 (NS). 50 ms ISI: WT n=11, hiw n= 9, hiw>dNmnat-RNAi n= 12. 1-way ANOVA w/Tukey's multiple comparisons, DF = 31, F= 5.587, p =0.0089. WT vs hiw p =0.0129 (*), hiw vs hiw> dNmnat-RNAi p= 0.0217 (*), WT vs hiw> dNmnat-RNAi p= 0.9546 (NS).]


Figure 4 ( A ) Representative images of NMJ synaptic terminals in control ( DVGlut - Gal4 > UAS - HA - dNmnat ) and hiw mutant ( hiwND9 , DVGlut - Gal4 > UAS - HA - dNmnat ) larvae . NMJs at larval muscle 4 were identified using HRP ( blue in top panel ) immunostaining . Insets highlight active zones within single boutons , which were identified by staining for the presynaptic active zone protein Brp ( red ) . An anti - HA antibody was used to stain for an HA - tagged dNmnat ( green ) transgene to measure protein levels in control and hiw mutant active zones . ( B ) Quantification of HA - dNmnat levels across the entire NMJ synaptic terminal , normalized to total terminal area and presented as fold change over WT . [ WT > HA - dNmnat n = 10 terminals , hiw > HA - dNmnat n = 10 terminals . Unpaired T - test , DF = 18 , p = 0 . 0028 ( * * ) ] . ( C ) Quantification of HA - dNmnat levels per unit area in WT and hiw for synaptic terminals excluding active zones ( herein referred to as " terminals " ) versus active zones ( AZs ) only . [ WT n = 10 terminals , hiw n = 9 terminals , each parsed into terminal minus AZs and AZs only . 2 - way ANOVA with Tukey ' s multiple comparisons , DF = 34 , p = 0 . 0005 for row factor ( terminal versus AZs ) and p < 0 . 0001 for column factor ( WT vs hiw ) . WT terminal vs hiw terminal p = 0 . 9830 ( NS ) . Hiw terminal vs hiw AZs p < 0 . 0001 ( * * * * ) . WT AZs vs hiw AZs p < 0 . 0001 ( * * * * ) . ]

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 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 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 4(A) Representative images of NMJ synaptic terminals in control (DVGlut-Gal4 > UAS-HA-dNmnat) and hiw mutant (hiwND9,DVGlut-Gal4 > UAS-HA-dNmnat) larvae. NMJs at larval muscle 4 were identified using HRP (blue in top panel) immunostaining. Insets highlight active zones within single boutons, which were identified by staining for the presynaptic active zone protein Brp (red). An anti-HA antibody was used to stain for an HA-tagged dNmnat (green) transgene to measure protein levels in control and hiw mutant active zones.(B) Quantification of HA-dNmnat levels across the entire NMJ synaptic terminal, normalized to total terminal area and presented as fold change over WT. [WT > HA-dNmnat n = 10 terminals, hiw > HA-dNmnat n = 10 terminals. Unpaired T-test, DF = 18, p = 0.0028 (**)].(C) Quantification of HA-dNmnat levels per unit area in WT and hiw for synaptic terminals excluding active zones (herein referred to as "terminals") versus active zones (AZs) only. [WT n = 10 terminals, hiw n = 9 terminals, each parsed into terminal minus AZs and AZs only. 2-way ANOVA with Tukey's multiple comparisons, DF = 34, p = 0.0005 for row factor (terminal versus AZs) and p < 0.0001 for column factor (WT vs hiw). WT terminal vs hiw terminal p = 0.9830 (NS). Hiw terminal vs hiw AZs p < 0.0001 (****). WT AZs vs hiw AZs p < 0.0001 (****).]