Figure 7 ( A , B ) Interaction of MYB with HIF1α ( A ) and p300 ( B ) was examined by co - immunoprecipitation assays in the lysates of pancreatic cancer cells cultured under hypoxia for 6 h . Immunoprecipitation with IgG isotype antibody was used as a control . 10 % of total cell lysate was used as input for western blot analysis . ( C , D ) Co - localization of MYB with HIF1α ( C ) and p300 ( D ) was examined in pancreatic cancer cells grown under hypoxia for 6 h by immunofluorescence assays followed by confocal microscopy . Scale bar , 30 μm ( lower left corner ) . Lower panels are digitally magnified views of the marked rectangles . For ease of visualization , a pseudo color ( purple ) was adopted for p300 staining .

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

Figure 7(A, B) Interaction of MYB with HIF1α (A) and p300 (B) was examined by co-immunoprecipitation assays in the lysates of pancreatic cancer cells cultured under hypoxia for 6 h. Immunoprecipitation with IgG isotype antibody was used as a control. 10% of total cell lysate was used as input for western blot analysis.(C, D) Co-localization of MYB with HIF1α (C) and p300 (D) was examined in pancreatic cancer cells grown under hypoxia for 6 h by immunofluorescence assays followed by confocal microscopy. Scale bar, 30 μm (lower left corner). Lower panels are digitally magnified views of the marked rectangles. For ease of visualization, a pseudo color (purple) was adopted for p300 staining.


Figure 8 ( B , C ) Tumor growth was monitored by non - invasive bioluminescence imaging . Tumors were resected at the experimental endpoint , and their weights ( B ) and volumes ( C ) were recorded . ( G ) Tumor xenograft serial sections were immunostained for pimonidazole ( PDZ ) adduct , MYB , HK2 , GLUT3 , and MCT4 using specific antibodies . Images were taken at 40X magnification . Scale bar , 60 μm ( lower left corner ) . The dashed lines demarcate the hypoxic region based on the PDZ staining pattern .

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Figure 8(B, C) Tumor growth was monitored by non-invasive bioluminescence imaging. Tumors were resected at the experimental endpoint, and their weights (B) and volumes (C) were recorded.(G) Tumor xenograft serial sections were immunostained for pimonidazole (PDZ) adduct, MYB, HK2, GLUT3, and MCT4 using specific antibodies. Images were taken at 40X magnification. Scale bar, 60 μm (lower left corner). The dashed lines demarcate the hypoxic region based on the PDZ staining pattern.


Figure 1A . U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes , at which point 4 mM HU was added and cells further incubated for the indicated times . Whole cell extracts were then analysed by western blotting with the indicated antibodies . Reduction of phosphorylation of Ser40 / 41 on MCM2 is indicative of CDC7 inhibition by XL413 . Representative of two independent experiments . B . U2OS cells were either mock treated or treated with 10 μM XL413 , 4 mM HU or both for 24 hours before performing neutral comet assays . Representative images of comets are shown . Scale bar = 100 μM . In the dot plots ~ 800 comets per each condition were analysed , means are indicated with a red line and their value shown above the plot . Data are from two independent experiments . C . U2OS cells were either untreated or treated with 10 μM XL413 or with 5 µM AZD6738 alone or in combination and , where indicated , after 30 minutes 4 mM HU was added for a further 2 hours . Whole cell extracts were analysed by western blotting with the indicated antibodies . Total protein stain ( TPS ) as a loading control . Representative of two independent experiments . D . U2OS cells were either untreated or treated with 4 mM HU for 5 hours in the presence or absence of 10 μM XL413 which had either been added to cells 30 minutes prior ( pre - treatment ) or at the same time ( co - treatment ) to the addition of HU . Whole cell extracts were analysed by western blotting with the indicated antibodies and total protein stain ( TPS ) is displayed as a loading control . Representative of two independent experiments .

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 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

Figure 1A. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells further incubated for the indicated times. Whole cell extracts were then analysed by western blotting with the indicated antibodies. Reduction of phosphorylation of Ser40/41 on MCM2 is indicative of CDC7 inhibition by XL413. Representative of two independent experiments.B. U2OS cells were either mock treated or treated with 10 μM XL413, 4 mM HU or both for 24 hours before performing neutral comet assays. Representative images of comets are shown. Scale bar = 100 μM. In the dot plots ~800 comets per each condition were analysed, means are indicated with a red line and their value shown above the plot. Data are from two independent experiments.C. U2OS cells were either untreated or treated with 10 μM XL413 or with 5 µM AZD6738 alone or in combination and, where indicated, after 30 minutes 4 mM HU was added for a further 2 hours. Whole cell extracts were analysed by western blotting with the indicated antibodies. Total protein stain (TPS) as a loading control. Representative of two independent experiments.D. U2OS cells were either untreated or treated with 4 mM HU for 5 hours in the presence or absence of 10 μM XL413 which had either been added to cells 30 minutes prior (pre-treatment) or at the same time (co-treatment) to the addition of HU. Whole cell extracts were analysed by western blotting with the indicated antibodies and total protein stain (TPS) is displayed as a loading control. Representative of two independent experiments.


Figure 2B . CSK soluble and chromatin - enriched protein fractions were analysed by western blotting with the indicated antibodies . Representative of two independent experiments . C - D . Flow cytometry analysis to assess levels of pS139 Histone H2AX ( c ) . Histograms show the mono - parametric analysis of cell count against pS139 Histone H2AX intensity . Histograms are overlaid to appreciate changes in pS139 Histone H2AX intensity upon treatment ( Red lines ) relative to appropriate experimental baseline controls ( Grey lines ) . Data are representative of two independent experiments .

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Figure 2B. CSK soluble and chromatin-enriched protein fractions were analysed by western blotting with the indicated antibodies. Representative of two independent experiments.C-D. Flow cytometry analysis to assess levels of pS139 Histone H2AX (c). Histograms show the mono-parametric analysis of cell count against pS139 Histone H2AX intensity. Histograms are overlaid to appreciate changes in pS139 Histone H2AX intensity upon treatment (Red lines) relative to appropriate experimental baseline controls (Grey lines). Data are representative of two independent experiments.


Figure 3A . U2OS cells were labelled with EdU for 30 minutes , then EdU was washed and cells were further incubated for either 1 or 2 hours in presence of thymidine . At the indicated time points cells were fixed and proteins binding to EdU - labelled DNA captured by DNA mediated chromatin Pull down technique ( DmChP ) . Graphical experimental outline is shown above the analysis by western blot of relevant proteins in both input and captured material . B . U2OS cells were labelled with EdU for 30 minutes and then treated with 4 mM HU for 2 hours in the presence or absence of 10 μM XL413 . Proteins binding to EdU - labelled DNA captured by DmChP were then analysed by western blot as above . C . As in panel B but incubation with HU was extended to 24 hours . Black arrow indicates MRE11 electrophoretic - mobility shift . D . U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes , at which point 4 mM HU was added and cells incubated for further 24 hours . Extracts prepared from HU treated cells were then incubated in presence or absence of λ - phosphatase . Proteins were analysed by western blotting with anti - MRE11 antibodies . Total protein stain ( TPS ) is used as a loading control . Black arrow indicates MRE11 electrophoretic - mobility shift . E . U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 hours . PCNA ( green ) and MRE11 ( red ) were detected by immunofluorescence . Inset I - II represent enlargements of selected region of the merged images . F . Quantification of PCNA and MRE11 colocalization was assessed with ImageJ in ~ 70 randomly selected cells for each condition from four biological replicates and expressed as Pearson ' s correlation coefficient . Error bars represent SEM . Statistical significance was assessed by student t test ( * p ˂ 0 .

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Figure 3A. U2OS cells were labelled with EdU for 30 minutes, then EdU was washed and cells were further incubated for either 1 or 2 hours in presence of thymidine. At the indicated time points cells were fixed and proteins binding to EdU-labelled DNA captured by DNA mediated chromatin Pull down technique (DmChP). Graphical experimental outline is shown above the analysis by western blot of relevant proteins in both input and captured material.B. U2OS cells were labelled with EdU for 30 minutes and then treated with 4 mM HU for 2 hours in the presence or absence of 10 μM XL413. Proteins binding to EdU-labelled DNA captured by DmChP were then analysed by western blot as above.C. As in panel B but incubation with HU was extended to 24 hours. Black arrow indicates MRE11 electrophoretic-mobility shift.D. U2OS cells were either mock or treated with 10 μM XL413 for 30 minutes, at which point 4 mM HU was added and cells incubated for further 24 hours. Extracts prepared from HU treated cells were then incubated in presence or absence of λ-phosphatase. Proteins were analysed by western blotting with anti-MRE11 antibodies. Total protein stain (TPS) is used as a loading control. Black arrow indicates MRE11 electrophoretic-mobility shift.E. U2OS cells were treated with 4 mM HU in the presence or absence of 10 μM XL413 for 24 hours. PCNA (green) and MRE11 (red) were detected by immunofluorescence. Inset I-II represent enlargements of selected region of the merged images.F. Quantification of PCNA and MRE11 colocalization was assessed with ImageJ in ~70 randomly selected cells for each condition from four biological replicates and expressed as Pearson's correlation coefficient. Error bars represent SEM. Statistical significance was assessed by student t test (*p˂0.05


Figure 4A . MCF10A cells were either mock or treated with 10 μM XL413 and labelled with IdU ( red ) for 30 minutes at which point 4 mM HU was added for 2 hours . HU was then washed off and cells labelled with CldU ( green ) in the continued presence or absence of XL413 . A set of representative DNA fibres from each condition is shown and the percentage of IdU ( red ) only tracts is plotted . At least 200 replication forks were analysed for each condition . Error bars represent the SEM from three biological repeats and statistical significance was assessed by the student t test ( * * p ˂ 0 . 01B . U2OS cells were either mock or treated with 10 μM XL413 , 50 μM Mirin or both , and labelled with IdU ( red ) for 30 minutes at which point 4 mM HU was added for 2 hours . HU was then washed off and cells labelled with CldU ( green ) in the continued presence or absence of XL413 and / or Mirin . A set of representative DNA fibres from each condition is shown and the percentage of IdU ( red ) only tracts is plotted . At least 200 replication forks were analysed for each condition . Error bars represent the SEM from four biological repeats and statistical significance was assessed by the student t test ( * p < 0 . 05 , * * p ˂ 0 . 01C . U2OS cells were either mock or pretreated for the indicated times with 10 μM XL413 , 50 μM Mirin or both , and labelled with CldU ( red ) . Then 50 nM CPT was added and cells labelled with of IdU ( green ) for further 30 minutes . A set of representative DNA fibres from each condition is shown and the IdU / CIdU tract length ratio is plotted . The box extends from the 25th to the 75th percentile with the line in the box representing the median . Whiskers indicate the 10 - 90 percentiles with data outside this range for individual outliers being plotted as dots . At least 100 replication forks were analyzed for each condition . P values were calculated using one way ANOVA Kruskal - Wallis test and Dunn ' s multiple comparison post test ( ns , not significant , * * * * p < 0 . 0001 ) and are related to the experiment shown . Similar results were obtained in a second independent experiment . D . U2OS cells were transfected with control ( siLuc ) or MRE11 targeting siRNA ( siMRE11 ) . After 48 hours cells were either mock or pretreated with 10 μM XL413 and labelled with CldU ( red ) for 30 minutes . Where indicated 50 nM CPT was added and then cells were labelled with IdU ( green ) . A set of representative DNA fibres from each condition is shown and the IdU / CIdU tract length ratio is plotted . The box extends from the 25th to the 75th percentile with the line in the box representing the median . Whiskers indicate the 10 - 90 percentileswith data outside this range for individual outliers being plotted as dots . At least 100 replication forks were analyzed for each condition . P values were calculated using one way ANOVA Kruskal - Wallis test and Dunn ' s multiple comparison post test ( ns , not significant , * * * * p < 0 . 0001 ) and are related to the experiment shown . Similar results were obtained in a second independent experiment .

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 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 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 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

Figure 4A. MCF10A cells were either mock or treated with 10 μM XL413 and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from three biological repeats and statistical significance was assessed by the student t test (** p˂0.01B. U2OS cells were either mock or treated with 10 μM XL413, 50 μM Mirin or both, and labelled with IdU (red) for 30 minutes at which point 4 mM HU was added for 2 hours. HU was then washed off and cells labelled with CldU (green) in the continued presence or absence of XL413 and/or Mirin. A set of representative DNA fibres from each condition is shown and the percentage of IdU (red) only tracts is plotted. At least 200 replication forks were analysed for each condition. Error bars represent the SEM from four biological repeats and statistical significance was assessed by the student t test (*p<0.05, ** p˂0.01C. U2OS cells were either mock or pretreated for the indicated times with 10 μM XL413, 50 μM Mirin or both, and labelled with CldU (red). Then 50 nM CPT was added and cells labelled with of IdU (green) for further 30 minutes. A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment.D. U2OS cells were transfected with control (siLuc) or MRE11 targeting siRNA (siMRE11). After 48 hours cells were either mock or pretreated with 10 μM XL413 and labelled with CldU (red) for 30 minutes. Where indicated 50 nM CPT was added and then cells were labelled with IdU (green). A set of representative DNA fibres from each condition is shown and the IdU/CIdU tract length ratio is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentileswith data outside this range for individual outliers being plotted as dots. At least 100 replication forks were analyzed for each condition. P values were calculated using one way ANOVA Kruskal-Wallis test and Dunn's multiple comparison post test (ns, not significant, **** p<0.0001) and are related to the experiment shown. Similar results were obtained in a second independent experiment.


Figure 5B . Representative western blot of indicated proteins obtained from whole cell extracts . pS40 / 41 on MCM2 indicates effective CDC7 inhibition by XL413 . C . Ratios of IdU / CldU tracts length is plotted . The box extends from the 25th to the 75th percentile with the line in the box representing the median . Whiskers indicate the 10 - 90 percentiles with data outside this range for individual outliers being plotted as dots . Mean values of IdU / CldU ratios are indicated above the plot . At least 100 replication forks were analyzed for each condition . D . The mean values of IdU / CldU tract length ratios from three independent experiments are plotted , with the standard deviation ( blue lines ) and mean ( red line ) . Statistical analysis : student t - test ; ns , not significant ; * * P < 0 . 01 , * * * P < 0 . 001E . Electron micrographs of representative replication forks from U2OS cells A regressed arm is labelled : P = parental strand ; D = daughter strand ; and R = reversed fork ; the four - way junction at the reversed fork is magnified in the inset . The red arrows indicates single stranded DNA at the fork . Scale bar is 200 nm ( = 460 bp ) , 100 nm ( = 230 bp ) in the inset . F . Frequency of reversed replication forks isolated from mock - depleted ( siLuc ) and BRCA2 - depleted ( siBRCA2 ) U2OS cells upon 5 hour treatment with 4 mM HU in the presence and absence of 10 μM XL413 . The number of replication intermediates analyzed is indicated in parentheses . Similar results were obtained in two independent experiments ( Appendix TableG . Amount of ssDNA length at the junction ( Red arrows in Figure 4D ) in siLuc and siBRCA2 U2OS cells treated with 4 mM of HU for 5 hours in the presence or absence of XL413 . N indicates the number of forks observed , and only molecules with detectable ssDNA stretches are included in the analysis . The lines show the mean length of ssDNA regions at the fork and the value is displayed above the

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

Figure 5B. Representative western blot of indicated proteins obtained from whole cell extracts. pS40/41 on MCM2 indicates effective CDC7 inhibition by XL413.C. Ratios of IdU/CldU tracts length is plotted. The box extends from the 25th to the 75th percentile with the line in the box representing the median. Whiskers indicate the 10-90 percentiles with data outside this range for individual outliers being plotted as dots. Mean values of IdU/CldU ratios are indicated above the plot. At least 100 replication forks were analyzed for each condition.D. The mean values of IdU/CldU tract length ratios from three independent experiments are plotted, with the standard deviation (blue lines) and mean (red line). Statistical analysis: student t-test; ns, not significant; **P<0.01, ***P < 0.001E. Electron micrographs of representative replication forks from U2OS cells A regressed arm is labelled: P=parental strand; D=daughter strand; and R=reversed fork; the four-way junction at the reversed fork is magnified in the inset. The red arrows indicates single stranded DNA at the fork. Scale bar is 200 nm ( = 460 bp), 100 nm ( = 230 bp) in the inset.F. Frequency of reversed replication forks isolated from mock-depleted (siLuc) and BRCA2-depleted (siBRCA2) U2OS cells upon 5 hour treatment with 4 mM HU in the presence and absence of 10 μM XL413. The number of replication intermediates analyzed is indicated in parentheses. Similar results were obtained in two independent experiments (Appendix TableG. Amount of ssDNA length at the junction (Red arrows in Figure 4D) in siLuc and siBRCA2 U2OS cells treated with 4 mM of HU for 5 hours in the presence or absence of XL413. N indicates the number of forks observed, and only molecules with detectable ssDNA stretches are included in the analysis. The lines show the mean length of ssDNA regions at the fork and the value is displayed above the plot


Figure 6A . Representative metaphase spread from BRCA2 depleted U2OS cell treated with HU . Scale bar = 5 μM . Arrows indicate the chromosomes enlarged in the insets , two of which show chromatid breaks ( * B - C . The graphs show the average number of chromatid breaks per spread . In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed . Error bars represent SEM . Statistical significance assessed by student t test ( * * p ˂ 0 . 01 , * * * p < 0 . 001D - E . U2OS and AS - CDC7 cells were transfected with control ( siCon ) or BRCA2 ( siBRCA2 ) targeting siRNAs . 48 hours post transfection cells were either mock - treated or treated with 10 μM XL413 or 10 μM 3MB - PP1 respectively for 24 hours with Nocodazole added for the last 16 hours of the experiment . The graph shows the average number of chromatid breaks per spread . In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed . Error bars represent SEM . Statistical significance assessed by student t test ( * p < 0 . 05 , * * * p < 0 .

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

Figure 6A. Representative metaphase spread from BRCA2 depleted U2OS cell treated with HU. Scale bar = 5 μM. Arrows indicate the chromosomes enlarged in the insets, two of which show chromatid breaks (*B-C. The graphs show the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (** p˂0.01, *** p<0.001D-E. U2OS and AS-CDC7 cells were transfected with control (siCon) or BRCA2 (siBRCA2) targeting siRNAs. 48 hours post transfection cells were either mock-treated or treated with 10 μM XL413 or 10 μM 3MB-PP1 respectively for 24 hours with Nocodazole added for the last 16 hours of the experiment. The graph shows the average number of chromatid breaks per spread. In each experiment 30 chromosome spreads for each condition were analysed and three independent experiments were performed. Error bars represent SEM. Statistical significance assessed by student t test (*p<0.05, *** p<0.001


Figure 3A . Hematoxylin and eosin ( H & E ) - stained cross section of a human liver slice . Scale bar is representative of A - F . Image is also used in Fig . S3A . B . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from A . The image displays a heatmap gradient of intensity from white ( least abundant ) to red ( most abundant ) which is representative of B , D , and F . C . H & E - stained cross section of a human lung slice . Image is also used in Fig . S3D . D . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from C . E . H & E - stained cross section of frontal cortical region of a human brain tissue . Image is also used in Fig . S3G . F . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) in an immediate adjacent tissue slice from E . G . Quantification of relative abundance of total glycogen calculated from the sum of CL3 - CL18 in human liver , lung , and brain . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * 0 . 01 < P < 0 . 05 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 , analyzed by one - way ANOVA with Tukey ' s multiple comparison for total glycogen . H . Glycogen structure represented by normalized chain length distribution ( ratio to CL3 ) of each human liver , lung and brain from G . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) .

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

Figure 3A. Hematoxylin and eosin (H&E)-stained cross section of a human liver slice. Scale bar is representative of A-F. Image is also used in Fig. S3A. B. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from A. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant) which is representative of B, D, and F. C. H&E-stained cross section of a human lung slice. Image is also used in Fig. S3D. D. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from C. E.H&E-stained cross section of frontal cortical region of a human brain tissue. Image is also used in Fig. S3G. F. Spatial distribution and relative abundance of glycogen (represented by CL7) in an immediate adjacent tissue slice from E. G. Quantification of relative abundance of total glycogen calculated from the sum of CL3-CL18 in human liver, lung, and brain. Values are presented as mean +/- standard error (n=3 technical replicates per region). *0.01 < P < 0.05; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison for total glycogen. H. Glycogen structure represented by normalized chain length distribution (ratio to CL3) of each human liver, lung and brain from G. Values are presented as mean +/- standard error (n=3 technical replicates per region).


Figure 4A . Hematoxylin and eosin ( H & E ) - stained cross section of adenocarcinoma of the prostate , lung squamous cell carcinoma ( LUSC ) , lung adenocarcinoma ( LUAD ) , and Ewing sarcoma ( ES ) of the abdomen tumor . Annotated regions are T - tumor , S - stroma , D / G - duct / glandular , and A - adipose tissue . Scale bar is representative of A and B . Images are also used in Fig . S4A , 5D and S6G . B . Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section shown in A . The image displays a heatmap gradient of intensity with white ( least abundant ) to red ( most abundant ) . Images are also used in Fig . S4E , 5D , and S6G . C . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length ( CL ) distribution from tumor regions shown in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) .

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

Figure 4A. Hematoxylin and eosin (H&E)-stained cross section of adenocarcinoma of the prostate, lung squamous cell carcinoma (LUSC), lung adenocarcinoma (LUAD), and Ewing sarcoma (ES) of the abdomen tumor. Annotated regions are T-tumor, S-stroma, D/G-duct/glandular, and A-adipose tissue. Scale bar is representative of A and B. Images are also used in Fig. S4A, 5D and S6G. B. Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Images are also used in Fig. S4E, 5D, and S6G. C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from tumor regions shown in A. Values are presented as mean +/- standard error (n=3 technical replicates per region).


Figure 5A . ( Left ) Hematoxylin and eosin ( H & E ) - stained cross section of an Ewing sarcoma ( ES ) of the shoulder tumor . Annotated regions are annotated as T - tumor and M - muscle . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . The image displays a heatmap gradient of intensity from white ( least abundant ) to red ( most abundant ) . Scale bar and intensity gradient below are representative of all images in A - E . B . ( Left ) H & E stained cross section of an ES of the chest wall tumor . Annotated regions are annotated as T - tumor and N - necrosis . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . C . ( Left ) H & E stained cross section of an ES of the rib tumor . Annotated regions are annotated as T - tumor and N - necrosis . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . D . ( Left ) H & E stained cross section of an ES of the abdomen tumor . Annotated regions are T - tumor , S - stroma , and A - adipose tissue . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . E . ( Left ) H & E stained cross section of an ES of the bladder tumor . Annotated regions are T - tumor and S - stroma . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section . F . Magnified H & E cross section of ( top , left ) tumor , and ( top , right ) muscle tissue from ES rib with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) tumor , and ( bottom , right ) muscle tissues . G . Magnified H & E cross section of ( top , left ) tumor , ( top , middle ) stroma , and ( top , right ) adipose tissues from ES abdomen with magnified image of CL7 ( m / z = 1175 ) of ( bottom , left ) tumor , ( bottom , middle ) stroma and ( bottom , right ) adipose tissues .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. (Left) Hematoxylin and eosin (H&E)-stained cross section of an Ewing sarcoma (ES) of the shoulder tumor. Annotated regions are annotated as T-tumor and M-muscle. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. The image displays a heatmap gradient of intensity from white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A-E. B. (Left) H&E stained cross section of an ES of the chest wall tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. C. (Left) H&E stained cross section of an ES of the rib tumor. Annotated regions are annotated as T-tumor and N-necrosis. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. D. (Left) H&E stained cross section of an ES of the abdomen tumor. Annotated regions are T-tumor, S-stroma, and A-adipose tissue. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. E. (Left) H&E stained cross section of an ES of the bladder tumor. Annotated regions are T-tumor and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section. F. Magnified H&E cross section of (top, left) tumor, and (top, right) muscle tissue from ES rib with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, and (bottom, right) muscle tissues. G. Magnified H&E cross section of (top, left) tumor, (top, middle) stroma, and (top, right) adipose tissues from ES abdomen with magnified image of CL7 (m/z=1175) of (bottom, left) tumor, (bottom, middle) stroma and (bottom, right) adipose tissues.


Figure 6A . ( Left ) Hematoxylin and eosin ( H & E ) stained cross section of a normal human tibia ( Top ) resected at the same time as the Ewing sarcoma ( ES ) of the tibia tumor ( Bottom ) . Annotated regions are CT - connective tissue , B - decalcified bone , BM - bone marrow , T - tumor , and S - stroma . ( Right ) Spatial distribution and relative abundance of glycogen ( represented by CL7 ) from an immediate adjacent resected tissue section shown in A . The image displays a heatmap gradient of intensity with white ( least abundant ) to red ( most abundant ) . Scale bar and intensity gradient below are representative of all images in A . B . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length ( CL ) distribution from normal tibia in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . # 0 . 01 < P < 0 . 05 ; * * * * P < 0 . 0001 , analyzed by two - way ANOVA with Tukey ' s multiple comparison for each glycogen chain length . C . Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length ( CL ) distribution from the ES of the tibia tumor in A . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * 0 . 001 < P < 0 . 01 ; * * * * P < 0 . 0001 , analyzed by two - tailed t - test for each glycogen chain length . F . In vivo tumor growth of A673 EV or GYS1 - KO injected in the flanks of athymic nude mice . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test . G . Half maximal effective concentration ( EC50 ) of the small molecular inhibitor guaiacol in A673 cells in vitro . J . Spatial distribution of phosphorylated CL5 of human tibia ( top ) and tibial ES tumor ( bottom ) . The image displays a heatmap gradient of intensity with black ( least abundant ) to yellow ( most abundant ) . K . Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J . Values are presented as mean + / - standard error ( n = 3 technical replicates per region ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test for each glycogen chain length . N . Representatives image of immunohistochemical staining of laforin in ES patient samples with annotation between tumor ( T ) and stroma ( S ) tissues . Tissues were scanned digitally using the Axio Scan . Z1 side scanner . Scale bar is shown below the image . O . Quantification of immunohistochemical laforin staining between normal tissue ( n = 3 technical replicates per region ) , tibial ES tumor ( n = 7 technical replicates per region ) , and stroma of five ES tissues ( n = 7 ) . Values are presented as mean + / - standard error . * * 0 . 001 < P < 0 . 0 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 , analyzed by one - way ANOVA with Tukey ' s multiple comparison . P . ( Top ) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors . ( Bottom ) Tumor growth of xenografts of A673 empty vector ( EV ) and laforin overexpression ( LAOE ) . Values are presented as mean + / - standard error ( n = 8 biological replicates per group ) . * * * * P < 0 . 0001 , analyzed by two - tailed t - test .

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

Figure 6A. (Left) Hematoxylin and eosin (H&E) stained cross section of a normal human tibia (Top) resected at the same time as the Ewing sarcoma (ES) of the tibia tumor (Bottom). Annotated regions are CT-connective tissue, B-decalcified bone, BM-bone marrow, T-tumor, and S-stroma. (Right) Spatial distribution and relative abundance of glycogen (represented by CL7) from an immediate adjacent resected tissue section shown in A. The image displays a heatmap gradient of intensity with white (least abundant) to red (most abundant). Scale bar and intensity gradient below are representative of all images in A. B. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from normal tibia in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). #0.01 < P < 0.05; ****P<0.0001, analyzed by two-way ANOVA with Tukey's multiple comparison for each glycogen chain length.C. Glycogen structure defined by distribution of released glucose polymers as representation of glycogen chain length (CL) distribution from the ES of the tibia tumor in A. Values are presented as mean +/- standard error (n=3 technical replicates per region). **0.001 < P < 0.01; ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.F. In vivo tumor growth of A673 EV or GYS1-KO injected in the flanks of athymic nude mice. Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.G. Half maximal effective concentration (EC50) of the small molecular inhibitor guaiacol in A673 cells in vitro.J. Spatial distribution of phosphorylated CL5 of human tibia (top) and tibial ES tumor (bottom). The image displays a heatmap gradient of intensity with black (least abundant) to yellow (most abundant). K. Phosphorylated chain length distribution in normal human tibia and tibial ES tumor in J. Values are presented as mean +/- standard error (n=3 technical replicates per region). ****P<0.0001, analyzed by two-tailed t-test for each glycogen chain length.N. Representatives image of immunohistochemical staining of laforin in ES patient samples with annotation between tumor (T) and stroma (S) tissues. Tissues were scanned digitally using the Axio Scan.Z1 side scanner. Scale bar is shown below the image. O. Quantification of immunohistochemical laforin staining between normal tissue (n=3 technical replicates per region), tibial ES tumor (n=7 technical replicates per region), and stroma of five ES tissues (n=7). Values are presented as mean +/- standard error. **0.001 < P < 0.0; ***P < 0.001; ****P<0.0001, analyzed by one-way ANOVA with Tukey's multiple comparison.P. (Top) Western blot showing confirmation of laforin overexpression in A673 xenograft tumors. (Bottom) Tumor growth of xenografts of A673 empty vector (EV) and laforin overexpression (LAOE). Values are presented as mean +/- standard error (n=8 biological replicates per group). ****P<0.0001, analyzed by two-tailed t-test.


Figure 1 ( B ) HEK293T cells transiently expressing FLAG and FLAG - tagged TEX264 were subjected to immunoprecipitation ( IP ) . Inputs ( 20 % of the lysates ) and immunoprecipitates ( from 80 % of the lysates ) were analyzed by immunoblotting using the indicated antibodies . ( C ) HEK293T cells transiently expressing WT or mutated TEX264 - FLAG were subjected to immunoprecipitation with an anti - FLAG antibody and detected with the indicated antibodies . ( D and E ) MEFs stably expressing TEX264 - GFP or its mutant were cultured in starvation media and immunostained with an anti - LC3 antibody . Bars : 10 µm and 1 µm ( insets ) ( D ) . Quantification of the number of TEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 48 cells for each cell type ( E ) . ( F and G ) WT and TEX264 - KO HeLa cells ( with or without the indicated TEX264 mutants with a C - terminal FLAG tag ) stably expressing the ER - phagy reporter ( ss - RFP - GFP - KDEL ) were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , cells were cultured in starvation medium lacking amino acids and serum for 9 h ( F ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± standard error of the mean ( SEM ) of three independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( G ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1(B) HEK293T cells transiently expressing FLAG and FLAG-tagged TEX264 were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies.(C) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.(D and E) MEFs stably expressing TEX264-GFP or its mutant were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 1 µm (insets) (D). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 48 cells for each cell type (E).(F and G) WT and TEX264-KO HeLa cells (with or without the indicated TEX264 mutants with a C-terminal FLAG tag) stably expressing the ER-phagy reporter (ss-RFP-GFP-KDEL) were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured in starvation medium lacking amino acids and serum for 9 h (F). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± standard error of the mean (SEM) of three independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (G).


Figure 2 ( A and B ) MEFs stably expressing TEX264 - GFP were cultured in starvation media with or without bafilomycin A1 ( Baf A1 ) and immunostained with anti - pTEX264 and anti - LC3 antibodies . Bars : 10 µm and 1 µm ( insets ) ( A ) . Quantification of the number of pTEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 35 cells for each cell type ( B ) . ( C ) HeLa cells were treated with 250 nM Torin 1 and Baf A1 for 16 h , and cell homogenates were subjected to an OptiPrep membrane flotation analysis . ( D , E and F ) WT , ATG3 - KO , and FIP200 - KO HeLa cells were cultured in starvation media lacking amino acids and serum with or without Baf A1 for 1 or 5 h . Cell lysates were analyzed by immunoblotting using the indicated antibodies ( D ) . Relative changes of the ratio of band intensities of phosphorylated TEX264 to HSP90 and total TEX264 during starvation are shown ( E and F ) . Data represent the mean ± SEM of three independent experiments .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) MEFs stably expressing TEX264-GFP were cultured in starvation media with or without bafilomycin A1 (Baf A1) and immunostained with anti-pTEX264 and anti-LC3 antibodies. Bars: 10 µm and 1 µm (insets) (A). Quantification of the number of pTEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 35 cells for each cell type (B).(C) HeLa cells were treated with 250 nM Torin 1 and Baf A1 for 16 h, and cell homogenates were subjected to an OptiPrep membrane flotation analysis.(D, E and F) WT, ATG3-KO, and FIP200-KO HeLa cells were cultured in starvation media lacking amino acids and serum with or without Baf A1 for 1 or 5 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies (D). Relative changes of the ratio of band intensities of phosphorylated TEX264 to HSP90 and total TEX264 during starvation are shown (E and F). Data represent the mean ± SEM of three independent experiments.


Figure 3 ( A ) HeLa cells were cultured with 20 nM CK1 inhibitor ( D4476 ) or CK2 inhibitor ( CX4945 ) for 3 h , followed by starvation ( with the inhibitors ) for 3 h . Cell lysates were analyzed by immunoblotting using the indicated antibodies . ( B ) Recombinant TEX264 - FLAG was incubated with or without CK2 and the CK2 inhibitor ( CX4945 ) in the presence of 200 µM ATP for 1 h . ( C ) HEK293T cells transiently expressing TEX264 - FLAG and one of the three Halo - CK2A isoforms or Halo - CK2B were subjected to immunoprecipitation with an anti - FLAG antibody and detected with anti - Halo and anti - FLAG antibodies . ( D and E ) HEK293T cells transiently expressing WT or TEX264 - FLAG mutants were subjected to immunoprecipitation ( IP ) . Inputs ( 20 % of the lysates ) and immunoprecipitates ( from 80 % of the lysates ) were analyzed by immunoblotting using the indicated antibodies ( D ) . The band intensities in the IP fractions were quantified , and the phospho - serine : FLAG ratio was calculated . Data represent the mean ± SEM of five independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( E ) . ( F and G ) MEFs stably expressing TEX264 - GFP were cultured with the CK2 inhibitor for 3 h followed by starvation ( with the CK2 inhibitor ) and immunostained with an anti - LC3 antibody . Scale bars represent 10 μm and 1 μm ( insets ) ( F ) . Quantification of the number of TEX264 puncta per cell . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by an unpaired two - tailed Student ' s t - test . Data were collected from 85 cells for each cell type ( G ) . ( H and I ) WT HeLa cells stably expressing the ER - phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , cells were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation ( with the CK2 inhibitor ) for 6 h ( H ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± SEM of six independent experiments . Differences were statistically analyzed by an unpaired two - tailed Student ' s t - test ( I ) .

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

Figure 3(A) HeLa cells were cultured with 20 nM CK1 inhibitor (D4476) or CK2 inhibitor (CX4945) for 3 h, followed by starvation (with the inhibitors) for 3 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies.(B) Recombinant TEX264-FLAG was incubated with or without CK2 and the CK2 inhibitor (CX4945) in the presence of 200 µM ATP for 1 h.(C) HEK293T cells transiently expressing TEX264-FLAG and one of the three Halo-CK2A isoforms or Halo-CK2B were subjected to immunoprecipitation with an anti-FLAG antibody and detected with anti-Halo and anti-FLAG antibodies.(D and E) HEK293T cells transiently expressing WT or TEX264-FLAG mutants were subjected to immunoprecipitation (IP). Inputs (20% of the lysates) and immunoprecipitates (from 80% of the lysates) were analyzed by immunoblotting using the indicated antibodies (D). The band intensities in the IP fractions were quantified, and the phospho-serine:FLAG ratio was calculated. Data represent the mean ± SEM of five independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (E).(F and G) MEFs stably expressing TEX264-GFP were cultured with the CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) and immunostained with an anti-LC3 antibody. Scale bars represent 10 μm and 1 μm (insets) (F). Quantification of the number of TEX264 puncta per cell. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by an unpaired two-tailed Student's t-test. Data were collected from 85 cells for each cell type (G).(H and I) WT HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, cells were cultured with 20 nM CK2 inhibitor for 3 h followed by starvation (with the CK2 inhibitor) for 6 h (H). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of six independent experiments. Differences were statistically analyzed by an unpaired two-tailed Student's t-test (I).


Figure 4 ( B ) HEK293T cells transiently expressing WT or mutated TEX264 - FLAG were subjected to immunoprecipitation with an anti - FLAG antibody and detected with the indicated antibodies . ( C and D ) MEFs stably expressing TEX264 - GFP or its mutants were cultured in starvation media and immunostained with an anti - LC3 antibody . Bars : 10 µm and 5 µm ( insets ) ( C ) . The intensity of the GFP signal under starvation conditions was quantified at TEX264 - GFP puncta and the ER . The puncta : ER signal ratio was calculated . Solid bars indicate the medians , boxes the interquartile range ( 25th to 75th percentile ) , and whiskers the 0th to 100th percentile . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test . Data were collected from 70 puncta for each cell type ( D ) . ( E and F ) WT and TEX264 - KO ( expressing WT or TEX264 - FLAG mutants ) HeLa cells stably expressing the ER - phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter . After doxycycline was removed , the cells were cultured in starvation medium lacking amino acids and serum for 9 h ( E ) . The band intensities of RFP and RFP - GFP were quantified and the ratio of RFP : RFP - GFP ( normalized to WT ) is shown . Data represent the mean ± SEM of four independent experiments . Differences were statistically analyzed by one - way ANOVA and Tukey ' s multiple comparison test ( F ) .

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

Figure 4(B) HEK293T cells transiently expressing WT or mutated TEX264-FLAG were subjected to immunoprecipitation with an anti-FLAG antibody and detected with the indicated antibodies.(C and D) MEFs stably expressing TEX264-GFP or its mutants were cultured in starvation media and immunostained with an anti-LC3 antibody. Bars: 10 µm and 5 µm (insets) (C). The intensity of the GFP signal under starvation conditions was quantified at TEX264-GFP puncta and the ER. The puncta:ER signal ratio was calculated. Solid bars indicate the medians, boxes the interquartile range (25th to 75th percentile), and whiskers the 0th to 100th percentile. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test. Data were collected from 70 puncta for each cell type (D).(E and F) WT and TEX264-KO (expressing WT or TEX264-FLAG mutants) HeLa cells stably expressing the ER-phagy reporter were cultured in the presence of doxycycline for 24 h to induce the reporter. After doxycycline was removed, the cells were cultured in starvation medium lacking amino acids and serum for 9 h (E). The band intensities of RFP and RFP-GFP were quantified and the ratio of RFP:RFP-GFP (normalized to WT) is shown. Data represent the mean ± SEM of four independent experiments. Differences were statistically analyzed by one-way ANOVA and Tukey's multiple comparison test (F).


Figure 1A Transfected GFP − Atg8 homologs were immunoprecipitated with μMACS ™ from total U2OS cell extracts followed by immunoblot analysis with anti − GFP and anti − ALFY antibodies . Data are representative of two independent experiments . C The indicated 35S − labelled in vitro − translated GFP − tagged constructs were incubated with GST , GST − LC3B or GST − GABARAP conjugated to glutathione Sepharose , and their binding was evaluated by autoradiography ( ARG ) . 5 % of the in vitro − translated protein ( arrow head ) used was loaded . Equal amounts of GST proteins were used as shown by Coomassie Brilliant Blue ( CBB ) staining . Data are representative of three independent experiments . D Recombinant ALFY ( aa 2981 - 3526 ) was incubated with GST ‐ Atg8 proteins conjugated to glutathione Sepharose . The pulled ‐ down complexes were subjected to SDS - PAGE and anti ‐ ALFY and anti ‐ GST immunoblotting . 5 % of the recombinant ALFY protein used was loaded as input . Data are representative of three independent experiments .

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Figure 1A Transfected GFP−Atg8 homologs were immunoprecipitated with μMACS™ from total U2OS cell extracts followed by immunoblot analysis with anti−GFP and anti−ALFY antibodies. Data are representative of two independent experiments.C The indicated 35S−labelled in vitro−translated GFP−tagged constructs were incubated with GST, GST−LC3B or GST−GABARAP conjugated to glutathione Sepharose, and their binding was evaluated by autoradiography (ARG). 5% of the in vitro−translated protein (arrow head) used was loaded. Equal amounts of GST proteins were used as shown by Coomassie Brilliant Blue (CBB) staining. Data are representative of three independent experiments.D Recombinant ALFY (aa 2981-3526) was incubated with GST‐Atg8 proteins conjugated to glutathione Sepharose. The pulled‐down complexes were subjected to SDS-PAGE and anti‐ALFY and anti‐GST immunoblotting. 5% of the recombinant ALFY protein used was loaded as input. Data are representative of three independent experiments.


Figure 2B 35S − labelled in vitro − translated GFP − ALFY ( aa 2981 - 3526 ) wild − type and different LIR mutants were incubated with GST − GABARAP or − LC3C and binding evaluated by ARG . 10 and 2 % of the in vitro − translated proteins used were loaded to illustrate binding affinity . CBB staining shows equal amounts of GST proteins used . Data are representative of three independent experiments . C MBP , MBP − ALFY ( aa 3255 - 3526 ) or the ALFY − LIR mutant ( F3346A ) conjugated to amylose resins was incubated in the absence or presence of purified GABARAP . The pulled − down complexes were subjected to SDS - PAGE and visualized by CBB staining . Data are representative of three independent experiments . D Thermodynamic parameters of ALFY − LIR peptide ( aa 3341 - 3354 ) to Atg8 homologs . All titrations were performed at 25 ° C as described in Supplementary Methods . ITC data were fitted to a one − site binding model . ND , not detected . E 35S − labelled in vitro − translated GFP or GFP − Bchs ( aa 3326 - 3341 ) were incubated with GST or GST − dAtg8a and binding evaluated by ARG . 5 % of the in vitro − translated protein used was loaded . Ponceau staining shows equal amounts of GST proteins used . Data are representative of three independent experiments .

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

Figure 2B 35S−labelled in vitro−translated GFP−ALFY (aa 2981-3526) wild−type and different LIR mutants were incubated with GST−GABARAP or −LC3C and binding evaluated by ARG. 10 and 2% of the in vitro−translated proteins used were loaded to illustrate binding affinity. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.C MBP, MBP−ALFY (aa 3255-3526) or the ALFY−LIR mutant (F3346A) conjugated to amylose resins was incubated in the absence or presence of purified GABARAP. The pulled−down complexes were subjected to SDS-PAGE and visualized by CBB staining. Data are representative of three independent experiments.D Thermodynamic parameters of ALFY−LIR peptide (aa 3341-3354) to Atg8 homologs. All titrations were performed at 25°C as described in Supplementary Methods. ITC data were fitted to a one−site binding model. ND, not detected.E 35S−labelled in vitro−translated GFP or GFP−Bchs (aa 3326-3341) were incubated with GST or GST−dAtg8a and binding evaluated by ARG. 5% of the in vitro−translated protein used was loaded. Ponceau staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.


Figure 3D GFP − LC3B and GFP − LC3B ( Q26K / H27Y / H57D ) were immunoprecipitated with GFP − TRAP ® from total cell lysate of stably transfected HeLa FlpIn cells , followed by SDS - PAGE and immunoblotting with the indicated antibodies . Data are representative of three independent experiments . E MBP ‐ ALFY3255 - 3526 conjugated to amylose resin was incubated with GST or the indicated GABARAP mutants . The pulled ‐ down complexes were subjected to SDS - PAGE and visualized by immunoblotting with anti ‐ MBP and anti ‐ GABARAP antibodies . Data are representative of three independent experiments . F MBP − tagged ALFY3255 - 3526 and p62168 - 391 or their corresponding LIR mutants ( F3346A and W340A , respectively ) were conjugated to amylose resin and incubated with purified GST , GABARAP , GABARAP mutant ( K24Q / Y25H / D54H ) , LC3B or LC3B mutant ( Q26K / H27Y / H57D ) . The bound proteins were visualized by immunoblotting with anti − GABARAP , anti − LC3 and anti − MBP antibodies . Data are representative of three independent experiments . G 35S ‐ labelled in vitro ‐ translated GFP ‐ ALFY ( aa 2981 - 3526 ) wild ‐ type and K3343A / D3344A / Y3351A mutant were incubated with GST ‐ LC3B and GABARAP and binding evaluated by ARG . 10 % of the in vitro ‐ translated proteins used was loaded . CBB staining shows equal amounts of GST proteins used . Data are representative of three independent experiments .

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Figure 3D GFP−LC3B and GFP−LC3B (Q26K/H27Y/H57D) were immunoprecipitated with GFP−TRAP® from total cell lysate of stably transfected HeLa FlpIn cells, followed by SDS-PAGE and immunoblotting with the indicated antibodies. Data are representative of three independent experiments.E MBP‐ALFY3255-3526 conjugated to amylose resin was incubated with GST or the indicated GABARAP mutants. The pulled‐down complexes were subjected to SDS-PAGE and visualized by immunoblotting with anti‐MBP and anti‐GABARAP antibodies. Data are representative of three independent experiments.F MBP−tagged ALFY3255-3526 and p62168-391 or their corresponding LIR mutants (F3346A and W340A, respectively) were conjugated to amylose resin and incubated with purified GST, GABARAP, GABARAP mutant (K24Q/Y25H/D54H), LC3B or LC3B mutant (Q26K/H27Y/H57D). The bound proteins were visualized by immunoblotting with anti−GABARAP, anti−LC3 and anti−MBP antibodies. Data are representative of three independent experiments.G 35S‐labelled in vitro‐translated GFP‐ALFY (aa 2981-3526) wild‐type and K3343A/D3344A/Y3351A mutant were incubated with GST‐LC3B and GABARAP and binding evaluated by ARG. 10% of the in vitro‐translated proteins used was loaded. CBB staining shows equal amounts of GST proteins used. Data are representative of three independent experiments.


Figure 4A HeLa FlpIn GFP − GABARAP cells were treated with proteasomal inhibitor ( MG132 , 2 h ) or subjected to amino acid starvation ( EBSS , 2 h ) , before staining with anti − ALFY antibodies . Scale bar , 10 μm . B GFP − tagged full − length ALFY wild − type or LC3 − interacting region ( LIR ) − mutant was expressed into Alfy − deficient MEFs using an adenovirus system . At 48 h after infection , the MEFs were cultured in normal media or EBSS for 1 . 5 h before staining with anti − LC3B or anti − GABARAP antibodies . Scale bars 10 μm . C HeLa cells were treated with control or siRNA targeting GABARAP , GABARAPL1 and GABARAPL2 . 72 h after transfection cells were immunostained with anti − LC3B , anti − ALFY and anti − p62 antibodies . Scale bars , 10 μm . D HeLa cells were treated with control or siRNA targeting ALFY . 72 h after transfection , cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX − 100 − soluble and insoluble fractions . The TX − 100 − soluble / insoluble fractions were then immunoblotted with the indicated antibodies . Data are representative of three independent experiments . E ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X − 100 ( TX − 100 ) − soluble and insoluble fractions . The TX − 100 − soluble / insoluble fractions were then immunoblotted with the indicated antibodies . Data are representative of three independent experiments .

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Figure 4A HeLa FlpIn GFP−GABARAP cells were treated with proteasomal inhibitor (MG132, 2 h) or subjected to amino acid starvation (EBSS, 2 h), before staining with anti−ALFY antibodies. Scale bar, 10 μm.B GFP−tagged full−length ALFY wild−type or LC3−interacting region (LIR)−mutant was expressed into Alfy−deficient MEFs using an adenovirus system. At 48 h after infection, the MEFs were cultured in normal media or EBSS for 1.5 h before staining with anti−LC3B or anti−GABARAP antibodies. Scale bars 10 μm.C HeLa cells were treated with control or siRNA targeting GABARAP, GABARAPL1 and GABARAPL2. 72 h after transfection cells were immunostained with anti−LC3B, anti−ALFY and anti−p62 antibodies. Scale bars, 10 μm.D HeLa cells were treated with control or siRNA targeting ALFY. 72 h after transfection, cells were treated with puromycin with or without bafilomycin A1 for 2 h and total cell lysates were fractionated into TX−100−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.E ALFY WT and KO MEFs were treated with puromycin or EBSS with or without bafilomycin A1 for 2 h and the total cell lysates were then fractionated into Triton X−100 (TX−100)−soluble and insoluble fractions. The TX−100−soluble/insoluble fractions were then immunoblotted with the indicated antibodies. Data are representative of three independent experiments.


Figure 1B . ERK71 mutants have elevated triacylglycerol ( TAG ) levels ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) . C . Fat body specific depletion of ERK7 by RNAi ( BDSC 56939 ) leads to increased TAG levels ( N = 4 replicates of 10 larvae / replicate for each genotype ) . D . Representative immunofluorescent images of lipid droplet ( LipidTOX ) and nuclear ( DAPI ) staining in control and ERK71 mutant fat bodies of third instar larvae . Scale bar : 50 µm . E . ERK71 mutant fat body cells contain more lipid droplets than control cells ( N = 30 cells for each genotype ) . F . After fed [ 13C ] glucose , the ERK71 mutant fat bodies display elevated [ 13C ] TAG / PE and unlabeled TAG / PE molar ratios ( measured by mass spectrometry - based lipidomics , N = 3 replicates of 15 fat bodies / replicate , TAG = triacylglycerol , PE = phosphatidylethanolamine ) G . ERK7 overexpression in the fat body results in reduced TAG levels , while kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence the TAG storage ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) . H - ERK7 expressing , GFP - marked , fat body clone ( H , marked by yellow dotted line ; scale bar : 50 µm ) visualized by LipidTOX staining ( N = 11 for control and 4 for ERK7 overexpression ) . ERK7 expressing , fat body clone contains less ( I ) and smaller ( J ) lipid droplets than control cells ( N = 11 for control and 4 for ERK7 overexpression ) . K . qRT - PCR based expression analysis of ERK7 from fat bodies of w1118 larvae upon 6 h starvation . Expression of RP49 was used for normalization ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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Figure 1B. ERK71 mutants have elevated triacylglycerol (TAG) levels (N=4 replicates of ≥10 larvae/ replicate for each genotype). C. Fat body specific depletion of ERK7 by RNAi (BDSC 56939) leads to increased TAG levels (N=4 replicates of 10 larvae/replicate for each genotype). D. Representative immunofluorescent images of lipid droplet (LipidTOX) and nuclear (DAPI) staining in control and ERK71 mutant fat bodies of third instar larvae. Scale bar: 50 µm. E. ERK71 mutant fat body cells contain more lipid droplets than control cells (N=30 cells for each genotype). F. After fed [13C]glucose, the ERK71 mutant fat bodies display elevated [13C]TAG/PE and unlabeled TAG/PE molar ratios (measured by mass spectrometry-based lipidomics, N=3 replicates of 15 fat bodies/replicate, TAG=triacylglycerol, PE=phosphatidylethanolamine)G. ERK7 overexpression in the fat body results in reduced TAG levels, while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the TAG storage (N=4 replicates of ≥10 larvae/replicate for each genotype).H- ERK7 expressing, GFP-marked, fat body clone (H, marked by yellow dotted line; scale bar: 50 µm) visualized by LipidTOX staining (N=11 for control and 4 for ERK7 overexpression).ERK7 expressing, fat body clone contains less (I) and smaller (J) lipid droplets than control cells (N=11 for control and 4 for ERK7 overexpression).K. qRT-PCR based expression analysis of ERK7 from fat bodies of w1118 larvae upon 6 h starvation. Expression of RP49 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).


Figure 2A . Fatty acid composition of total lipids in CG > control and CG > ERK7 larvae , measured by gas chromatography ( N = 3 replicates of 15 larvae / replicate for each genotype ) . C . Amount of glucose - derived [ 13C ] - labeled carbons incorporated into the TAG pool of control and CG > ERK7 fat bodies ( N = 3 replicates of 15 fat bodies / replicate for each genotype and diet ) . D . Ectopic ERK7 expression in the fat body results in elevated hemolymph glucose levels ( N = 3 replicates of 15 larvae / replicate for each genotype ) . E . Circulating glucose levels are decreased in ERK71 mutants ( N = 4 replicates of 15 larvae / replicate for each genotype ) .

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Figure 2A. Fatty acid composition of total lipids in CG>control and CG>ERK7 larvae, measured by gas chromatography (N=3 replicates of 15 larvae/replicate for each genotype).C. Amount of glucose-derived [13C]-labeled carbons incorporated into the TAG pool of control and CG>ERK7 fat bodies (N=3 replicates of 15 fat bodies/replicate for each genotype and diet).D. Ectopic ERK7 expression in the fat body results in elevated hemolymph glucose levels (N=3 replicates of 15 larvae/replicate for each genotype). E. Circulating glucose levels are decreased in ERK71 mutants (N=4 replicates of 15 larvae/replicate for each genotype).


Figure 3A . Four - way plot ( t - statistics ) presenting the ERK7 - dependent fasting - responsive genes ( adj . p . val < 0 . 05 ) . Dark blue indicates significant in both comparisons ; dark turquoise , significant in one comparison ; light turquoise , not significant . D . Heatmaps displaying expression patterns of selected genes involved in lipid anabolism and lipid catabolism . Color key displays scaled log2 gene expression . E , F . Expression analysis of Seipin , Agpat3 , ATPCL , fabp , Lip4 and CG34448 by qRT - PCR . RP49 was used for normalization ( N = 3 replicates of ≥ 10 larvae / replicate for each genotype ) .

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Figure 3A. Four-way plot (t-statistics) presenting the ERK7-dependent fasting-responsive genes (adj.p.val < 0.05). Dark blue indicates significant in both comparisons; dark turquoise, significant in one comparison; light turquoise, not significant.D. Heatmaps displaying expression patterns of selected genes involved in lipid anabolism and lipid catabolism. Color key displays scaled log2 gene expression.E, F. Expression analysis of Seipin, Agpat3, ATPCL, fabp, Lip4 and CG34448 by qRT-PCR. RP49 was used for normalization (N=3 replicates of ≥10 larvae/replicate for each genotype).


Figure 4A . Weight of control and ERK71 mutant larvae at 72h after egg deposition ( AED ) ( N = 4 replicates of 12 larvae / replicate for each genotype ) . B . Pupariation kinetics of control and ERK71 mutants ( N = 4 replicates of 30 larvae / replicate for each genotype ) . p < 0 . 0001 , analyzed by Log - rank test . C . ERK71 mutants fail to suppress growth upon 24 h of starvation ( N ≥ 4 replicates of 15 larvae / replicate for each genotype ) . D . ERK71 mutant larvae ( N = 4 replicates of 24 larvae / replicate for each genotype , p < 0 . 01 analyzed by Log - rank test ) are sensitive to nutrient deprivation . Fat body specific knockdown of ERK7 by RNAi ( BDSC 56939 ) leads to a moderately accelerated pupariation rate ( E , p < 0 . 001 analyzed by Log - rank test , N = 8 replicates of 30 larvae / replicate for each genotype ) F . Fat body specific knockdown of ERK7 by RNAi ( BDSC 56939 ) leads to an increase in larval weight at 72h AED ( F , N = 5 replicates of 15 larvae / replicate for each genotype ) . G . A representative image showing that fat body - specific overexpression of ERK7 by the CG - GAL4 driver ( CG > ERK7 ) impairs larval growth . Scale bar : 0 . 5 mm . H . Ectopic expression of ERK7 leads to delayed pupariation ( p < 0 . 0001 , analyzed by Log - rank test ) , while kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence pupariation rate ( N = 4 replicates of 30 larvae / replicate for each genotype ) . I . In contrast to ERK7 wt , kinase - dead ( K54R ) and activation loop phosphorylation - deficient ( T190A / Y192F ) mutants of ERK7 do not influence the pupal volume ( N = 4 replicates of 10 pupae / replicate for each genotype ) .

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Figure 4A. Weight of control and ERK71 mutant larvae at 72h after egg deposition (AED) (N=4 replicates of 12 larvae/replicate for each genotype).B. Pupariation kinetics of control and ERK71 mutants (N=4 replicates of 30 larvae/replicate for each genotype). p<0.0001, analyzed by Log-rank test.C. ERK71 mutants fail to suppress growth upon 24 h of starvation (N≥4 replicates of 15 larvae/replicate for each genotype).D. ERK71 mutant larvae (N=4 replicates of 24 larvae/replicate for each genotype, p<0.01 analyzed by Log-rank test) are sensitive to nutrient deprivation.Fat body specific knockdown of ERK7 by RNAi (BDSC 56939) leads to a moderately accelerated pupariation rate (E, p<0.001 analyzed by Log-rank test, N=8 replicates of 30 larvae/replicate for each genotype)F. Fat body specific knockdown of ERK7 by RNAi (BDSC 56939) leads to an increase in larval weight at 72h AED (F, N=5 replicates of 15 larvae/replicate for each genotype).G. A representative image showing that fat body-specific overexpression of ERK7 by the CG-GAL4 driver (CG>ERK7) impairs larval growth. Scale bar: 0.5 mm.H. Ectopic expression of ERK7 leads to delayed pupariation (p<0.0001, analyzed by Log-rank test), while kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence pupariation rate (N=4 replicates of 30 larvae/replicate for each genotype).I. In contrast to ERK7 wt, kinase-dead (K54R) and activation loop phosphorylation-deficient (T190A/Y192F) mutants of ERK7 do not influence the pupal volume (N=4 replicates of 10 pupae/replicate for each genotype).


Figure 5A . Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus . Scale bar : 30 µm . B , C . ERK71 mutant fat bodies display increased nuclear area ( B , N > 160 cells , obtained from 5 independent fat bodies ) and increased nucleolar / nuclear area ratio ( C , N > 80 cells for nucleolus / nucleus ratio , obtained from 5 independent fat bodies ) , when compared to control fat bodies . D - F . ERK7 expressing fat body clones ( D , marked by GFP , scale bar : 20 µm ) display reduced nuclear area ( E ) and reduced nucleolar / nuclear area ratio ( F ) , when compared to control cells . Nuclei were visualized by DAPI staining , nucleoli were visualized by anti - Fibrillarin antibodies ( N = 10 ) . G , H . Expression analysis of Pol I ( G ) and Pol III ( H ) target RNAs by qRT - PCR . Expression of CDK7 was used for normalization ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) .

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Figure 5A. Representative immunofluorescent images of control and ERK71 mutant fat bodies with DAPI staining to visualize nucleus. Scale bar: 30 µm. B, C. ERK71 mutant fat bodies display increased nuclear area (B, N>160 cells, obtained from 5 independent fat bodies) and increased nucleolar/nuclear area ratio (C, N>80 cells for nucleolus/nucleus ratio, obtained from 5 independent fat bodies), when compared to control fat bodies.D-F. ERK7 expressing fat body clones (D, marked by GFP, scale bar: 20 µm) display reduced nuclear area (E) and reduced nucleolar/nuclear area ratio (F), when compared to control cells. Nuclei were visualized by DAPI staining, nucleoli were visualized by anti-Fibrillarin antibodies (N=10).G, H. Expression analysis of Pol I (G) and Pol III (H) target RNAs by qRT-PCR. Expression of CDK7 was used for normalization (N=3 replicates of 10 fat bodies/replicate for each genotype).


Figure 6A . ERK7 overexpression leads to phosphorylation of PWP1 , as shown by appearance of an extra band on Phos - tag Western blot ( marked with red arrow ) . Insulin - inducible bands ( dark blue ) remain unresponsive to ERK7 . B . Representative immunofluorescent images of PWP1 localization in fat bodies of ERK71 mutant and control third instar larvae . Scale bar : 20 µm . C . PWP1 localization displays increased nucleolar / nuclear ratio in ERK71 mutant fat bodies compared to control ( N = 20 cells from 5 independent fat bodies per genotype ) . D . Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae . ERK7 overexpression ( GFP marked clones ) alters the subcellular localization of PWP1 . Scale bar : 30 µm . E . PWP1 localization displays reduced nuclear / cytoplasmic ratio in ERK7 overexpressing clones , compared to control cells ( N = 10 cells from at least 8 different fat bodies per genotype ) . F . Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) . G . Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body ( CG - GAL4 ) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus , respectively . Scale bar : 50 µm . H , I . Fat body specific knockdown of PWP1 suppresses increased lipid droplet number ( H ; N > 30 cells per genotype ) and increased nuclear area ( I ; N > 45 cells per genotype ) caused by ERK7 knockdown ( BDSC 56939 ) .

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Figure 6A. ERK7 overexpression leads to phosphorylation of PWP1, as shown by appearance of an extra band on Phos-tag Western blot (marked with red arrow). Insulin-inducible bands (dark blue) remain unresponsive to ERK7.B. Representative immunofluorescent images of PWP1 localization in fat bodies of ERK71 mutant and control third instar larvae. Scale bar: 20 µm. C. PWP1 localization displays increased nucleolar/nuclear ratio in ERK71 mutant fat bodies compared to control (N=20 cells from 5 independent fat bodies per genotype). D. Representative immunofluorescent images of PWP1 localization in fat bodies of third instar larvae. ERK7 overexpression (GFP marked clones) alters the subcellular localization of PWP1. Scale bar: 30 µm. E. PWP1 localization displays reduced nuclear/cytoplasmic ratio in ERK7 overexpressing clones, compared to control cells (N=10 cells from at least 8 different fat bodies per genotype). F. Fat body specific depletion of PWP1 by RNAi leads to decreased TAG levels (N=4 replicates of ≥10 larvae/replicate for each genotype).G. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK7 and PWP1 fat body (CG-GAL4) knockdown with LipidTOX staining and DAPI to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. H, I. Fat body specific knockdown of PWP1 suppresses increased lipid droplet number (H; N>30 cells per genotype) and increased nuclear area (I; N>45 cells per genotype) caused by ERK7 knockdown (BDSC 56939).


Figure 7A , B . Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 ( A ) and ectopic expression of ERK7 ( B ) ( N = 3 replicates of 10 fat bodies / replicate for each genotype ) . C . ERK71 mutants fail to maximally downregulate sugarbabe expression upon fasting ( N = 4 replicates of 10 larvae / replicate for each genotype ) . D . Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants ( sug17Δ / Df ( 2R ) Exel7123 ) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus , respectively . Scale bar : 50 µm . E . sug mutant ( sug17Δ / Df ( 2R ) Exel7123 ) suppresses increased lipid droplet number observed in ERK71 mutants ( N > 20 ) . F . Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG > ERK7 larvae ( N = 4 replicates of ≥ 10 larvae / replicate for each genotype ) . G . sug mutant ( sug17Δ / Df ( 2R ) Exel7123 ) suppresses increased nuclear size phenotype of ERK71 mutant fat body cells ( N ≥ 25 cells per genotype ) . Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate ( H ) ( p < 0 . 001 for the rescue , analyzed by Log - rank test , N = 4 replicates of 30 larvae / replicate for each genotype ) of CG > ERK7 larvaeI . Ectopic Sugarbabe expression in the fat body partially restores the pupal volume of CG > ERK7 larvae ( I ) ( N = 4 replicates of 10 pupae / replicate for each genotype ) . J . Addition of 20 % sucrose improves the growth rate and survival of CG > ERK7 larvae . Pupariation kinetics of CG > ERK7 larvae on 20 % yeast or 20 % yeast + 20 % sucrose diet ( p < 0 . 0001 for the rescue , analyzed by Log - rank test , N = 4 replicates of 30 larvae / replicate for each genotype and diet ) .

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Figure 7A, B. Expression of sugarbabe is downregulated in fat bodies upon knockdown of PWP1 (A) and ectopic expression of ERK7 (B) (N=3 replicates of 10 fat bodies/replicate for each genotype).C. ERK71 mutants fail to maximally downregulate sugarbabe expression upon fasting (N=4 replicates of 10 larvae/replicate for each genotype).D. Representative immunofluorescent images of fat bodies in a genetic epistasis experiment using ERK71 and sug mutants (sug17Δ/Df(2R)Exel7123) with LipidTOX and DAPI staining to visualize lipid droplets and nucleus, respectively. Scale bar: 50 µm. E. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased lipid droplet number observed in ERK71 mutants (N>20). F. Ectopic sugarbabe expression in the fat body partially rescues TAG levels in the CG>ERK7 larvae (N=4 replicates of ≥10 larvae/replicate for each genotype).G. sug mutant (sug17Δ/Df(2R)Exel7123) suppresses increased nuclear size phenotype of ERK71 mutant fat body cells (N ≥25 cells per genotype).Ectopic Sugarbabe expression in the fat body partially restores the pupariation rate (H) (p<0.001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype) of CG>ERK7 larvaeI. Ectopic Sugarbabe expression in the fat body partially restores the pupal volume of CG>ERK7 larvae (I) (N=4 replicates of 10 pupae/replicate for each genotype).J. Addition of 20% sucrose improves the growth rate and survival of CG>ERK7 larvae. Pupariation kinetics of CG>ERK7 larvae on 20% yeast or 20% yeast + 20% sucrose diet (p<0.0001 for the rescue, analyzed by Log-rank test, N=4 replicates of 30 larvae/replicate for each genotype and diet).


Figure 2 ( F ) Representative images of GFP - positive HEK293 cells at day 7 post - transduction using 1x106 TU / cell of RGD4C . TPA . GFP or RGD4C . AAVP . GFP particles . Non - targeted ( NT ) particles were included as control . Data shown are representative of five independent experiments , performed each in triplicates . Scale bar , 100 μm .

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Figure 2(F) Representative images of GFP-positive HEK293 cells at day 7 post-transduction using 1x106 TU/cell of RGD4C.TPA.GFP or RGD4C.AAVP.GFP particles. Non-targeted (NT) particles were included as control. Data shown are representative of five independent experiments, performed each in triplicates. Scale bar, 100 μm.