Figure 3Representative Coomassie - stained SDS - PAGE gel showing the isolation of SDS insoluble aggregates from mouse brain lysates . TH = total homogenate , SUP = supernatant , W1 = SDS - soluble fraction , Pellet = formic - acid soluble fraction ( see Figure EV4A ) . Quantification of the yield of SDS - insoluble aggregates from young and old brain lysates was based on densitometry analysis of Coomassie - stained gel bands obtained from different animals , n = 5 per age group ( Figure EV4B ) ; * * p < 0 . 01 , unpaired t - test . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range . Volcano plot based on protein quantification by label - free mass spectrometry depicting the enrichment of specific proteins in protein aggregates . The x - axis indicates the log2 ratio between protein abundance in aggregates ( Pellet ) and starting total homogenate ( TH ) . The horizontal dashed line indicates a p value cut - off of 0 . 05 and vertical lines a log2 fold change cut off of ± 0 . 5 . Selected proteins are highlighted as colored dots as indicated in the figure legend . Protein quantification was based on samples obtained from 3 independent isolations . Double - labelling of telencephalic sections of N . furzeri with anti - RPS6 ( green ) as ribosomal marker and Proteostat as a marker for aggregated proteins ( red ) . Nuclear counterstaining was performed with DAPI ( blue ) . Scale bar = 10 μm .

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Figure 3Representative Coomassie-stained SDS-PAGE gel showing the isolation of SDS insoluble aggregates from mouse brain lysates. TH = total homogenate, SUP = supernatant, W1 = SDS-soluble fraction, Pellet = formic-acid soluble fraction (see Figure EV4A). Quantification of the yield of SDS-insoluble aggregates from young and old brain lysates was based on densitometry analysis of Coomassie-stained gel bands obtained from different animals, n=5 per age group (Figure EV4B); ** p < 0.01, unpaired t-test. In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.Volcano plot based on protein quantification by label-free mass spectrometry depicting the enrichment of specific proteins in protein aggregates. The x-axis indicates the log2 ratio between protein abundance in aggregates (Pellet) and starting total homogenate (TH). The horizontal dashed line indicates a p value cut-off of 0.05 and vertical lines a log2 fold change cut off of ± 0.5. Selected proteins are highlighted as colored dots as indicated in the figure legend. Protein quantification was based on samples obtained from 3 independent isolations.Double-labelling of telencephalic sections of N. furzeri with anti-RPS6 (green) as ribosomal marker and Proteostat as a marker for aggregated proteins (red). Nuclear counterstaining was performed with DAPI (blue). Scale bar = 10 μm.


Figure 4Abundance changes of proteasome proteins and their transcripts during aging . Members of the 19S and 20S complex are displayed separately and changes are shown for both the age comparisons as box plots . Transcripts are displayed as light blue and proteins as dark blue boxes . Changes of individual proteins are displayed as dots , orange dots represent significant cases ( adj . p < 0 . 05 , n = 4 per age group for proteome and n = 5 per age group for transcriptome ) . In boxplots , the horizontal line represents the median , the bottom and top of the box the 25th and 75th percentile , respectively , and the whiskers extend 1 . 5 folds the interquartile range . Heat map showing transcript and protein fold changes for members of the 26 proteasome ( 19S and 12S complexes ) . Genes are annotated according to significance of their changes at the level of transcript ( adj . p < 0 . 05 : black , adj . p > 0 . 05 : white ) or protein ( adj . p < 0 . 05 : green , adj . p > 0 . 05 : white ) . In - gel proteasome assay following native gel electrophoresis ( top ) and immunoblotting of proteasome complexes ( 30S , 26S and 20S ) ( bottom ) in young ( 5wph ) , adult ( 12 wph ) and old ( 39 wph ) killifish brains . For additional samples and low exposure pictures see Figure EV5B and EV5C . Bar plots depicting the quantification of chymotrypsin - like ( CT - L ) activity from native gels calculated for doubly capped ( 30S ) or singly capped ( 26S ) proteasomes . n > = 5 per sample group ; error bars indicate standard error of the mean . * p < 0 . 05 , * * p < 0 . 005 , * * * p = 0 . 0001 , * * * * p < 0 . 001 , one - way ANOVA , Holm - Sidak ' s multiple comparison test . For each sample group , the mean value of activity in young samples ( 5 wph ) was set to 100 % . Percentage ( % ) of chymotrypsin - like ( CT - L ) , trypsin - like ( T - L ) and peptidylglutamyl peptide hydrolysing or caspase - like ( PGPH ) proteasome activities in brain extracts of killifish of different ages . n > = 6 per sample group ; error bars indicate standard error of the mean . * * p < 0 . 005 , * * * p = 0 . 0001 , * * * * p < 0 . 001 , one - way ANOVA , Holm - Sidak ' s multiple comparison test . For each sample group , the mean value of each activity in young samples ( 5 wph ) was set to 100 % . Age - related changes of proteins involved in the ubiquitin cycle . All the displayed proteins showed significant protein level changes in the 39 wph vs . 12 wph comparison ( adj . 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 4Abundance changes of proteasome proteins and their transcripts during aging. Members of the 19S and 20S complex are displayed separately and changes are shown for both the age comparisons as box plots. Transcripts are displayed as light blue and proteins as dark blue boxes. Changes of individual proteins are displayed as dots, orange dots represent significant cases (adj. p < 0.05, n=4 per age group for proteome and n=5 per age group for transcriptome). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.Heat map showing transcript and protein fold changes for members of the 26 proteasome (19S and 12S complexes). Genes are annotated according to significance of their changes at the level of transcript (adj. p < 0.05: black, adj. p > 0.05: white) or protein (adj. p < 0.05: green, adj. p > 0.05: white).In-gel proteasome assay following native gel electrophoresis (top) and immunoblotting of proteasome complexes (30S, 26S and 20S) (bottom) in young (5wph), adult (12 wph) and old (39 wph) killifish brains. For additional samples and low exposure pictures see Figure EV5B and EV5C.Bar plots depicting the quantification of chymotrypsin-like (CT-L) activity from native gels calculated for doubly capped (30S) or singly capped (26S) proteasomes. n >=5 per sample group; error bars indicate standard error of the mean. * p < 0.05, ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of activity in young samples (5 wph) was set to 100%.Percentage (%) of chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolysing or caspase-like (PGPH) proteasome activities in brain extracts of killifish of different ages. n >=6 per sample group; error bars indicate standard error of the mean. ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of each activity in young samples (5 wph) was set to 100%.Age-related changes of proteins involved in the ubiquitin cycle. All the displayed proteins showed significant protein level changes in the 39 wph vs. 12 wph comparison (adj. p < 0.05).


Figure 5Chymotrypsin - like ( CT - L ) proteasome activity in brain extracts of killifish treated with different proteasome inhibitors . The activity was measured at day 4 after the beginning of treatment . n = 8 per sample group ; error bars indicate standard deviation . * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA followed by Holm - Sidak ' s multiple comparisons test . Proteasome inhibition induces proteasome activators ( PSME4 ) , assembly factors ( POMP ) ubiquitin ligases ( UBE3C ) , mediators of autophagosome formation ( ATG2B ) , heat shock proteins and chaperones in killifish brain . Protein abundances were quantified by DIA mass spectrometry and they are shown relative to the mean value of vehicle control samples ( DMSO ) set to 1 , n = 10 per sample group . Adj . p < 0 . 05 for all the displayed proteins . Error bars indicate mean + / - SD . Bortezomib treatment affects the stoichiometry of a subset of protein complexes . The number of affected proteins ( adj . p < 0 . 25 ) for each protein complex is indicated . Only protein complexes that had at least two members affected are shown . Members of the mitochondrial and cytoplasmic ribosomes affected by Bortezomib treatment . Relative protein abundances ( normalized to the mean of the protein complex to which they belong ) are shown . The mean value of vehicle control samples ( DMSO ) was set to 1 . n = 10 per sample group . Adj . p < 0 . 25 for all the displayed proteins . Error bars indicate mean + / - SD .

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Figure 5Chymotrypsin-like (CT-L) proteasome activity in brain extracts of killifish treated with different proteasome inhibitors. The activity was measured at day 4 after the beginning of treatment. n=8 per sample group; error bars indicate standard deviation. ** p < 0.01, **** p < 0.0001, one-way ANOVA followed by Holm-Sidak's multiple comparisons test.Proteasome inhibition induces proteasome activators (PSME4), assembly factors (POMP) ubiquitin ligases (UBE3C), mediators of autophagosome formation (ATG2B), heat shock proteins and chaperones in killifish brain. Protein abundances were quantified by DIA mass spectrometry and they are shown relative to the mean value of vehicle control samples (DMSO) set to 1, n=10 per sample group. Adj. p < 0.05 for all the displayed proteins. Error bars indicate mean +/-SD.Bortezomib treatment affects the stoichiometry of a subset of protein complexes. The number of affected proteins (adj. p < 0.25) for each protein complex is indicated. Only protein complexes that had at least two members affected are shown.Members of the mitochondrial and cytoplasmic ribosomes affected by Bortezomib treatment. Relative protein abundances (normalized to the mean of the protein complex to which they belong) are shown. The mean value of vehicle control samples (DMSO) was set to 1. n=10 per sample group. Adj. p < 0.25 for all the displayed proteins. Error bars indicate mean +/-SD.


Figure 6Distribution of change in expression for proteasome transcripts across the entire cohort of 159 fish . Lifespan was compared among fish that showed extreme changes in proteasome levels between 10 and 20 wph ( 32 fish showing the most pronounced decreases , shown in black vs . 32 fish showing the most pronounce increases ) . * * * p < 0 . 001 Log - rank test .

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Figure 6Distribution of change in expression for proteasome transcripts across the entire cohort of 159 fish. Lifespan was compared among fish that showed extreme changes in proteasome levels between 10 and 20 wph (32 fish showing the most pronounced decreases, shown in black vs. 32 fish showing the most pronounce increases). *** p<0.001 Log-rank test.


Figure 1Percentage of 2 - cell - like cells obtained after hyperosmotic treatments for 24h ( B ) Shown are the mean ± s . d . from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Unpaired two - tailed t - tests were performed . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . Percentage of 2 - cell - like cells obtained after hyperosmotic treatments the indicated time ( C - D ) . Shown are the mean ± s . d . from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Unpaired two - tailed t - tests were performed . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . H - I . Protein levels assessed by Western blotting in the indicated conditions . Shown is one representative image from 2 independent replicates .

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Figure 1Percentage of 2-cell-like cells obtained after hyperosmotic treatments for 24h (B) Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.Percentage of 2-cell-like cells obtained after hyperosmotic treatments the indicated time (C-D). Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.H-I. Protein levels assessed by Western blotting in the indicated conditions. Shown is one representative image from 2 independent replicates.


Figure 2A . ( Left ) Representative single confocal z - section of CellROX - DeepRed fluorescence of ESC treated with 0 . 2M sorbitol for the indicated time from 2 biological replicates . Scale bar : 10µm . ( Right ) Quantification of CellROX - DeepRed fluorescence intensity from single z - sections from 2 biological replicates . Two - tailed Mann - Whitney tests were performed . Boxes indicate the range between the first and third quartile , the line indicates the median and the whiskers display the spread from 5 to 95 % of the data . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . B . Percentage of 2CLCs obtained by FACS after the indicated sorbitol treatment with addition of ROS scavenger N - AcetylCysteine ( NAC ) during either the sorbitol treatment or recovery time . Shown are the mean ± s . d . from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Unpaired two - tailed t - tests were performed . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . C . Protein levels assessed by Western blotting in the indicated conditions . Shown is one representative image from 3 independent replicates . D . Percentage of 2CLCs obtained by FACS after the indicated hyperosmotic treatment with addition of ATR inhibitor during either the hyperosmotic stress or recovery time . Shown are the mean ± s . d . from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Same untreated dots are shown . Two - tail t - tests were performed . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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 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 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

Figure 2A. (Left) Representative single confocal z-section of CellROX-DeepRed fluorescence of ESC treated with 0.2M sorbitol for the indicated time from 2 biological replicates. Scale bar: 10µm. (Right) Quantification of CellROX-DeepRed fluorescence intensity from single z-sections from 2 biological replicates. Two-tailed Mann-Whitney tests were performed. Boxes indicate the range between the first and third quartile, the line indicates the median and the whiskers display the spread from 5 to 95% of the data. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.B. Percentage of 2CLCs obtained by FACS after the indicated sorbitol treatment with addition of ROS scavenger N-AcetylCysteine (NAC) during either the sorbitol treatment or recovery time. Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.C. Protein levels assessed by Western blotting in the indicated conditions. Shown is one representative image from 3 independent replicates.D. Percentage of 2CLCs obtained by FACS after the indicated hyperosmotic treatment with addition of ATR inhibitor during either the hyperosmotic stress or recovery time. Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Same untreated dots are shown. Two-tail t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.


Figure 3A . Percentage of 2CLCs obtained by FACS after the indicated sorbitol treatment with addition of ROS scavenger N - AcetylCysteine ( NAC ) or ATR inhibitor . Shown are the mean ± s . d from 5 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Unpaired two - tailed t - tests were performed . B . Percentage of 2CLCs obtained by FACS after the indicated NaCl treatment with the addition of ROS scavenger N - AcetylCysteine ( NAC ) or ATR inhibitor . Shown are the mean ± s . d . from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Unpaired two - tailed t - tests were performed . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . C . Percentage of 2CLCs obtained by FACS after the indicated treatments . Shown are the mean ± s . d from at least 2 replicates . Individual dots indicate measurement of an independent biological replicate by FACS . Individual and combinatorial treatments performed with 0 . 2M of sorbitol , 24mM of sodium acetate , 2 . 5nM of PlaB spliceosome inhibitor and 0 . 53µM of retinoic acid . Unpaired two - tailed t - tests were performed . Unpaired two - tailed t - tests were performed against each individual treatment in the combinatorial experiments . Data information : Stars indicate significant differences obtained using indicated statistical tests with * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 .

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 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

Figure 3A. Percentage of 2CLCs obtained by FACS after the indicated sorbitol treatment with addition of ROS scavenger N-AcetylCysteine (NAC) or ATR inhibitor. Shown are the mean ± s.d from 5 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. B. Percentage of 2CLCs obtained by FACS after the indicated NaCl treatment with the addition of ROS scavenger N-AcetylCysteine (NAC) or ATR inhibitor. Shown are the mean ± s.d. from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Unpaired two-tailed t-tests were performed. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.C. Percentage of 2CLCs obtained by FACS after the indicated treatments. Shown are the mean ± s.d from at least 2 replicates. Individual dots indicate measurement of an independent biological replicate by FACS. Individual and combinatorial treatments performed with 0.2M of sorbitol, 24mM of sodium acetate, 2.5nM of PlaB spliceosome inhibitor and 0.53µM of retinoic acid. Unpaired two-tailed t-tests were performed. Unpaired two-tailed t-tests were performed against each individual treatment in the combinatorial experiments. Data information: Stars indicate significant differences obtained using indicated statistical tests with *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.


Figure 1 D . Time course of pyrene actin polymerization showing the influence of different SPIN90 constructs in reactions containing 50 nM Bos taurus Arp2 / 3 complex and 3 μM 15 % pyrene labeled actin . E . Plot of maximum polymerization rate versus concentration of SPIN90 for reactions shown in ( D ) F . Plot of maximum polymerization rate versus concentration of SPIN90 for pyrene actin polymerization assays containing 2 . 5 μM SPIN90 , 500 nM Arp2 / 3 complex , 3 μM actin and the indicated concentration of the SPIN90 ARM domain construct G . Time courses of pyrene actin polymerization as described in panel ( D ) , except the influence of the SPIN90 middle segment is tested on its own or in the presence of the 351 - 722 construct

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Figure 1 D. Time course of pyrene actin polymerization showing the influence of different SPIN90 constructs in reactions containing 50 nM Bos taurus Arp2/3 complex and 3 μM 15% pyrene labeled actin. E. Plot of maximum polymerization rate versus concentration of SPIN90 for reactions shown in (D)  F. Plot of maximum polymerization rate versus concentration of SPIN90 for pyrene actin polymerization assays containing 2.5 μM SPIN90, 500 nM Arp2/3 complex, 3 μM actin and the indicated concentration of the SPIN90 ARM domain construct  G. Time courses of pyrene actin polymerization as described in panel (D), except the influence of the SPIN90 middle segment is tested on its own or in the presence of the 351-722 construct


Figure 4 B . Time courses of pyrene actin polymerization showing the influence of mutations on the ability of SPIN90 ( 269 - 722 ) to activate Arp2 / 3 complex . Reactions contained 3 μM 15 % pyrene labeled actin , 50 nM Bos taurus Arp2 / 3 complex , and 20 μM wild type or mutant SPIN90 ( 269 - 722 ) . C . Plot of maximum polymerization rate versus concentration of SPIN90 ( 269 - 722 ) for reactions as shown in panel ( B )

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Figure 4 B. Time courses of pyrene actin polymerization showing the influence of mutations on the ability of SPIN90(269-722) to activate Arp2/3 complex. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex, and 20 μM wild type or mutant SPIN90(269-722). C. Plot of maximum polymerization rate versus concentration of SPIN90(269-722) for reactions as shown in panel (B)


Figure 5 A . Plot of maximum polymerization rate versus concentration of the small molecule Arp2 / 3 complex inhibitor CK - 666 . Reactions contained 3 μM 15 % pyrene labeled actin , 50 nM Bos taurus Arp2 / 3 complex and the indicated concentrations of CK - 666

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Figure 5 A. Plot of maximum polymerization rate versus concentration of the small molecule Arp2/3 complex inhibitor CK-666. Reactions contained 3 μM 15% pyrene labeled actin, 50 nM Bos taurus Arp2/3 complex and the indicated concentrations of CK-666


Figure 6B . SDS - PAGE gel showing results of actin filament copelleting assay . Actin filaments at 3 . 5 μM were used to co - pellet 0 . 6 μM Bos taurus Arp2 / 3 complex in the presence or absence of the indicated concentrations of SPIN90 ( 269 - 722 ) ( S : Soluble fraction , P : Pellet ) C . Plot of co - pelleting data as shown in ( B ) , run in quadruplicate with error bars showing the standard

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

Figure 6B. SDS-PAGE gel showing results of actin filament copelleting assay. Actin filaments at 3.5 μM were used to co-pellet 0.6 μM Bos taurus Arp2/3 complex in the presence or absence of the indicated concentrations of SPIN90(269-722) (S: Soluble fraction, P: Pellet) C. Plot of co-pelleting data as shown in (B), run in quadruplicate with error bars showing the standard deviation


Figure 1Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA - mediated p97 depletion under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( A ) ( * * * P < 0 . 001 ; unpaired t - test , n = 3 , mean + SEM ) . Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition ( CB5083 , 10µM for 6h ) under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( C ) ( * P < 0 . 05 , * * * * P < 0 . 0001 ; unpaired t - test , n = 3 , mean + SEM ) . Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline - induced mild expression of the p97EQ variant under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( E ) ( * * P < 0 . 01 , * * * * P < 0 . 0001 ; unpaired t - test , n = 4 , mean + SEM ) . Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA - mediated p97 depletion . Graph represents the quantifications of ( H ) ( * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean + SEM ) and Western blot for efficacy of siRNA depletion of p97 ( right ) . Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing K48 - linked hyper - ubiquitination of RNF8 after siRNA - mediated p97 depletion . Western blot analysis of Strep - p97 Co - IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97WT under physiological conditions and after IR ( 10 Gy ) . Graph represents the quantifications of ( K ) ( * P < 0 . 05 ; unpaired t - test , n = 2 , mean + SEM ) .

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 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 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 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

Figure 1Western blot analysis showing increased RNF8 protein level in HeLa cells after siRNA-mediated p97 depletion under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (A) (***P<0.001; unpaired t-test, n=3, mean +SEM).   Western blot analysis showing increased RNF8 protein level in HeLa cells after p97 chemical inhibition (CB5083, 10µM for 6h) under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (C) (*P<0.05, ****P<0.0001; unpaired t-test, n=3, mean +SEM).   Western blot analysis showing increased RNF8 protein level in HEK293 cells after doxycycline-induced mild expression of the p97EQ variant under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (E) (**P<0.01, ****P<0.0001; unpaired t-test, n=4, mean +SEM).   Western blot analysis of CHX chase kinetics showing reduced RNF8 degradation rate in HEK293 cells after siRNA-mediated p97 depletion.   Graph represents the quantifications of (H) (***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean +SEM) and Western blot for efficacy of siRNA depletion of p97 (right).   Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing K48-linked hyper-ubiquitination of RNF8 after siRNA-mediated p97 depletion.Western blot analysis of Strep-p97 Co-IP in HEK293 cells showing increased RNF8 interaction with p97EQ variant as compared to p97WT under physiological conditions and after IR (10 Gy).   Graph represents the quantifications of (K) (*P<0.05; unpaired t-test, n=2, mean +SEM).


Figure 2Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing the ubiquitination pattern of RNF8 - WT and RNF8 - RING * variant , under siRNA - mediated luciferase ( siLuc ) or p97 - depleted conditions ( sip97 ) . Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition ( MG132 , 10µM ) . Graph represents the quantifications of ( B ) ( ns ; not significant , P > 0 . 05 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM ) . Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing hyper - ubiquitination of RNF8 after proteasome inhibition ( MG132 , 10µM for 6h ) . Western blot analysis of CHX chase kinetics in U2OS cells , comparing the degradation rate of Flag - RNF8 - WT and Flag - RNF8 - RING * . Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8 . Graph represents the quantifications of ( E ) ( ns P > 0 . 05 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean + SEM ) .

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 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

Figure 2Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing the ubiquitination pattern of RNF8-WT and RNF8-RING* variant, under siRNA-mediated luciferase (siLuc) or p97-depleted conditions (sip97).Western blot analysis of CHX chase kinetics in HeLa cells showing the degradation kinetics of RNF8 and inhibition of RNF8 degradation by simultaneous proteasome inhibition (MG132, 10µM).   Graph represents the quantifications of (B) (ns; not significant, P>0.05, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).   Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing hyper-ubiquitination of RNF8 after proteasome inhibition (MG132, 10µM for 6h).Western blot analysis of CHX chase kinetics in U2OS cells, comparing the degradation rate of Flag-RNF8-WT and Flag-RNF8-RING*. Endogenous RNF8 was depleted by shRNF8 targeting only endogenous RNF8.   Graph represents the quantifications of (E) (ns P>0.05, ****P<0.0001; two-way ANOVA, n=3, mean +SEM).


Figure 3Western blot analysis of Strep - p97 Co - IP in nuclear fraction of HEK293 cells showing interaction of p97 with ATX3 under physiological conditions and after IR ( 10 Gy ) . Western blot analysis of Flag - ATX3 Co - IP in HEK293 cells showing interaction of ATX3 with p97 , RNF8 and PSα6 under physiological conditions and after IR ( 10 Gy ) . Western blot analysis of Flag - ATX3 WT or Flag - ATX3 VBM * Co - IP in HEK293 cells showing interaction of ATX3 with p97 , RNF8 and p97 core co - factors Npl4 - Ufd1 . ( * represents unspecific bands ) . Western blot analysis of Strep - p97EQ Co - IP in HEK293 cells , in presence or absence ( CRISPR knockout ; ∆ ATX3 ) of ATX3 , showing increased interaction of RNF8 with p97 in absence of ATX3 , under physiological conditions and after IR ( 10 Gy ) . Graph represents quantifications of ( F ) ( * P < 0 . 05 , * * P < 0 . 01 ; unpaired t - test , n = 3 , mean + SEM ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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 3Western blot analysis of Strep-p97 Co-IP in nuclear fraction of HEK293 cells showing interaction of p97 with ATX3 under physiological conditions and after IR (10 Gy).Western blot analysis of Flag-ATX3 Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and PSα6 under physiological conditions and after IR (10 Gy).Western blot analysis of Flag-ATX3 WT or Flag-ATX3 VBM* Co-IP in HEK293 cells showing interaction of ATX3 with p97, RNF8 and p97 core co-factors Npl4-Ufd1. (* represents unspecific bands).Western blot analysis of Strep-p97EQ Co-IP in HEK293 cells, in presence or absence (CRISPR knockout; ∆ATX3) of ATX3, showing increased interaction of RNF8 with p97 in absence of ATX3, under physiological conditions and after IR (10 Gy).   Graph represents quantifications of (F) (*P<0.05, **P<0.01; unpaired t-test, n=3, mean +SEM).


Figure 4Western blot analysis of CHX chase kinetics in HeLa cells showing accelerated endogenous RNF8 degradation in the soluble fraction ( Cytosol and Nucleosol ) of ∆ ATX3 cell extract . Arrow represents the main RNF8 band and asterisks represents unspecific bands . Graphs represent the quantifications of ( A ) . RNF8 level at starting point ( 0h ) was shown without equalisation ( left ) . In order to nullify the difference in RNF8 level at starting point ( 0h ) , we equalised RNF8 level to 100 % and then compared the degradation rate ( right ) . ( * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM ) . Western blot analysis of CHX chase showing the kinetics of endogenous RNF8 degradation in the soluble fraction ( Cytosol and Nucleosol ) of ATX3 knockdown HeLa cells . The degradation was completely blocked after simultaneous inhibition of proteasome ( MG132 , 10µM ) . Western blot analysis of Flag - RNF8 denaturing - IP in HEK293 cells showing RNF8 hyper - ubiquitination in siRNA - mediated p97 or ATX3 - depleted conditions . Western blot analysis of Flag - RNF8 denaturing - IP in HeLa cells showing RNF8 hyper - ubiquitination in ∆ ATX3 condition . RNF8 hyper - ubiquitination was suppressed by reintroduction of GFP - ATX3 - WT but not with GFP - ATX3 - C14A or GFP - ATX3 - UIM * .

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

Figure 4Western blot analysis of CHX chase kinetics in HeLa cells showing accelerated endogenous RNF8 degradation in the soluble fraction (Cytosol and Nucleosol) of ∆ATX3 cell extract. Arrow represents the main RNF8 band and asterisks represents unspecific bands.   Graphs represent the quantifications of (A). RNF8 level at starting point (0h) was shown without equalisation (left). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate (right). (**P<0.01, ***P<0.001, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).   Western blot analysis of CHX chase showing the kinetics of endogenous RNF8 degradation in the soluble fraction (Cytosol and Nucleosol) of ATX3 knockdown HeLa cells. The degradation was completely blocked after simultaneous inhibition of proteasome (MG132, 10µM).Western blot analysis of Flag-RNF8 denaturing-IP in HEK293 cells showing RNF8 hyper-ubiquitination in siRNA-mediated p97 or ATX3-depleted conditions.Western blot analysis of Flag-RNF8 denaturing-IP in HeLa cells showing RNF8 hyper-ubiquitination in ∆ATX3 condition. RNF8 hyper-ubiquitination was suppressed by reintroduction of GFP-ATX3-WT but not with GFP-ATX3-C14A or GFP-ATX3-UIM*.


Figure 5Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction ( cytoplasm + nucleoplasm ) of HeLa cells , expressing DOX - inducible GFP - ATX3 - C14A variant as compared to GFP - ATX3 - WT . Endogenous ATX3 was depleted with siRNA targeting 3 ' UTR region of ATXN3 . Graph represents the quantifications of ( A ) . In order to nullify the difference in RNF8 level at starting point ( 0h ) , we equalised RNF8 level to 100 % and then compared the degradation rate . RNF8 level at starting point ( 0h ) was also shown without equalisation . ( * P < 0 . 05 , * * P < 0 . 01 ; two - way ANOVA , n = 2 , mean + SEM ) . Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction ( cytoplasm + nucleoplasm ) of HeLa cells , expressing DOX - inducible ATX3 - UIM * variant as compared to ATX3 - WT . Endogenous ATX3 was depleted with siRNA ATX3 _ 3 ' UTR . Graph represents the quantifications of ( C ) . In order to nullify the difference in RNF8 level at starting point ( 0h ) , we equalised RNF8 level to 100 % and then compared the degradation rate . RNF8 level at starting point ( 0h ) was also shown without equalisation . ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM ) . Western blot analysis showing endogenous RNF8 protein level in soluble fraction ( Cytosol and Nucleosol ) of HeLa cells under indicated conditions . RNF8 level was significantly reduced after siRNA mediated ATX3 depletion and then was significantly rescued by DOX - inducible expression of GFP - ATX3 - WT but not with GFP - ATX3 - VBM . Graphs represent the quantifications of ( E ) . ( ns P > 0 . 05 , * P < 0 . 05 , * * P < 0 . 01 ; unpaired t - test , n = 3 , mean + SEM ) . Representative IF images showing γH2AX - positive micronuclei ( white arrows ) under physiological conditions with indicated siRNA mediated depletion . ( Scale bar represents 10 µm ) Graph represents quantification of ( G ) , showing percentage of cells with γH2AX - positive micronuclei in U2OS cells . ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; unpaired t - test , n = 3 , mean + SEM , at least 200 randomly selected nuclei were counted per condition per 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm+nucleoplasm) of HeLa cells, expressing DOX-inducible GFP-ATX3-C14A variant as compared to GFP-ATX3-WT. Endogenous ATX3 was depleted with siRNA targeting 3'UTR region of ATXN3.   Graph represents the quantifications of (A). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0h) was also shown without equalisation. (*P<0.05, **P<0.01; two-way ANOVA, n=2, mean +SEM).   Western blot analysis of CHX chase showing accelerated degradation of RNF8 in soluble fraction (cytoplasm + nucleoplasm) of HeLa cells, expressing DOX-inducible ATX3-UIM* variant as compared to ATX3-WT. Endogenous ATX3 was depleted with siRNA ATX3_3'UTR.   Graph represents the quantifications of (C). In order to nullify the difference in RNF8 level at starting point (0h), we equalised RNF8 level to 100% and then compared the degradation rate. RNF8 level at starting point (0h) was also shown without equalisation. (ns P>0.05, *P<0.05, **P<0.01, ****P<0.0001; two-way ANOVA, n=2, mean +SEM).   Western blot analysis showing endogenous RNF8 protein level in soluble fraction (Cytosol and Nucleosol) of HeLa cells under indicated conditions. RNF8 level was significantly reduced after siRNA mediated ATX3 depletion and then was significantly rescued by DOX-inducible expression of GFP-ATX3-WT but not with GFP-ATX3-VBM.   Graphs represent the quantifications of (E). (ns P>0.05, * P<0.05, **P<0.01; unpaired t-test, n=3, mean +SEM).   Representative IF images showing γH2AX-positive micronuclei (white arrows) under physiological conditions with indicated siRNA mediated depletion. (Scale bar represents 10 µm)   Graph represents quantification of (G), showing percentage of cells with γH2AX-positive micronuclei in U2OS cells. (* P<0.05, **P<0.01, ***P<0.001; unpaired t-test, n=3, mean +SEM, at least 200 randomly selected nuclei were counted per condition per experiment).


Figure 6Quantifications of neutral comet assay showing the level of DSBs under indicated siRNA - depleted conditions , before and after IR ( 4 Gy ) . ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean + SEM ) . Colony forming assay ( CFA ) showing the IR sensitivity of HeLa cells under siRNA - mediated p97 or ATX3 - depleted conditions . ( n = 3 , mean ± SD ) . CFA showing the IR sensitivity of HeLa - WT cells compared to HeLa ∆ ATX3 cells . ( n = 3 , mean ± SD ) . CFA showing the rescue of cell survival in IR treated , ATX3 - depleted ( siRNA ) , HeLa cells , after DOX - inducible expression of ATX3 - WT but not with ATX3 - C14A or ATX3 - UIM * variant . ( ns P > 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean ± SD ) . CFA showing the rescue of cell survival in IR treated , ATX3 - depleted ( siRNA ) , HeLa cells , after DOX - inducible expression of ATX3 - WT but not with ATX3 - VBM variant . ( ns P > 0 . 05 , * P < 0 . 05 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean ± SD ) . Representative IF micrographs in U2OS cells showing IRIF formation of GFP - RNF8 - WT , 53BP1 and γH2AX ( first panel ) and GFP - RNF8 - ∆ RING , 53BP1 and γH2AX ( second panel ) under indicated siRNA - depleted or p97 - inhibited ( CB5083 ) conditions . ( Scale bar ; 10µm ) . Quantification of ( F ) for GFP - RNF8 - WT . Graph represents the percentage of cells with increased GFP - RNF8 - WT foci ( white bars ) or percentage of cells with > 5 53BP1 foci ( grey bars ) . ( * * * P < 0 . 001 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM , at least 100 nuclei per condition and experiment ) . Quantification of ( F ) for GFP - RNF8 - ∆ RING . Graph represents the percentage of cells with increased GFP - RNF8 - ∆ RING foci ( white bars ) or percentage of cells with > 5 53BP1 foci ( grey bars ) . ( ns P > 0 . 05 , * P < 0 . 01 , * * P < 0 . 01 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 2 , mean + SEM , at least 100 nuclei per condition and experiment ) . Representative IF images and quantification showing endogenous K63 - Ub and γ - H2AX at UV - A micro - laser - induced DNA damage tracks in U2OS cells under indicated siRNA - depleted conditions . ( Scale bar ; 10µm ) . Quantification of ( I ) . Graph represents the average intensity of the K63 - Ub ( ns P > 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; unpaired t - test , n = 2 , mean + SEM , more than 50 nuclei were analysed ) .

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 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 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 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 6Quantifications of neutral comet assay showing the level of DSBs under indicated siRNA-depleted conditions, before and after IR (4 Gy). (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean +SEM).Colony forming assay (CFA) showing the IR sensitivity of HeLa cells under siRNA-mediated p97 or ATX3-depleted conditions. (n=3, mean ±SD).   CFA showing the IR sensitivity of HeLa-WT cells compared to HeLa∆ATX3 cells. (n=3, mean ±SD).   CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-C14A or ATX3-UIM* variant. (ns P>0.05, **P<0.01, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).   CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after DOX-inducible expression of ATX3-WT but not with ATX3-VBM variant. (ns P>0.05, *P<0.05, ***P<0.001, ****P<0.0001; two-way ANOVA, n=3, mean ±SD).   Representative IF micrographs in U2OS cells showing IRIF formation of GFP-RNF8-WT, 53BP1 and γH2AX (first panel) and GFP-RNF8-∆RING, 53BP1 and γH2AX (second panel) under indicated siRNA-depleted or p97-inhibited (CB5083) conditions. (Scale bar; 10µm).   Quantification of (F) for GFP-RNF8-WT. Graph represents the percentage of cells with increased GFP-RNF8-WT foci (white bars) or percentage of cells with >5 53BP1 foci (grey bars). (***P<0.001, ****P<0.0001; two-way ANOVA, n=2, mean +SEM, at least 100 nuclei per condition and experiment).   Quantification of (F) for GFP-RNF8-∆RING. Graph represents the percentage of cells with increased GFP-RNF8-∆RING foci (white bars) or percentage of cells with >5 53BP1 foci (grey bars). (ns P>0.05, *P<0.01, **P<0.01, ****P<0.0001; two-way ANOVA, n=2, mean +SEM, at least 100 nuclei per condition and experiment).   Representative IF images and quantification showing endogenous K63-Ub and γ-H2AX at UV-A micro-laser-induced DNA damage tracks in U2OS cells under indicated siRNA-depleted conditions. (Scale bar; 10µm).   Quantification of (I). Graph represents the average intensity of the K63-Ub (ns P>0.05, **P<0.01, ***P<0.001; unpaired t-test, n=2, mean +SEM, more than 50 nuclei were analysed).


Figure 7Representative IF images showing the average intensity of endogenous RNF168 at UV - A micro - laser - induced DNA damage tracks in U2OS cells . ( Scale bar ; 10µm ) . Graph represents the quantification of ( A ) . ( * * * P < 0 . 001 ; unpaired t - test , n = 3 , mean + SEM , more than 50 nuclei were analysed ) . Representative IF images showing the average intensity of endogenous 53BP1 at UV - A micro - laser - induced DNA damage tracks in U2OS cells . ( Scale bar ; 10µm ) . Graph represents the quantification of ( C ) . ( ns P > 0 . 05 , * * * P < 0 . 001 ; unpaired t - test , n = 2 , mean + SEM , at least 80 nuclei per condition and experiment ) . Western blot analysis of Flag - histone H1 denaturing - IP , showing ubiquitination pattern of histone H1 after 30 min of IR ( 10 Gy ) treatment under indicated siRNA - depleted conditions . Representative IF images showing the average intensity of endogenous BRCA1 at UV - A micro - laser - induced DNA damage tracks in U2OS cells . ( Scale bar ; 10µm ) . Graph represents the quantification of ( F ) . ( ns P > 0 . 05 , * * * P < 0 . 001 ; unpaired t - test , n = 2 , mean + SEM , at least 100 nuclei per condition and 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative IF images showing the average intensity of endogenous RNF168 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (A). (***P<0.001; unpaired t-test, n=3, mean +SEM, more than 50 nuclei were analysed).   Representative IF images showing the average intensity of endogenous 53BP1 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (C). (ns P>0.05, ***P<0.001; unpaired t-test, n=2, mean +SEM, at least 80 nuclei per condition and experiment).   Western blot analysis of Flag-histone H1 denaturing-IP, showing ubiquitination pattern of histone H1 after 30 min of IR (10 Gy) treatment under indicated siRNA-depleted conditions.Representative IF images showing the average intensity of endogenous BRCA1 at UV-A micro-laser-induced DNA damage tracks in U2OS cells. (Scale bar; 10µm).   Graph represents the quantification of (F). (ns P>0.05, ***P<0.001; unpaired t-test, n=2, mean +SEM, at least 100 nuclei per condition and experiment).


Figure 8GFP based reporter assay showing the efficiency of NHEJ repair pathway under indicated siRNA - depleted conditions in HEK293 cells . Graph represents the quantifications from three independent experiments ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ; one - way ANOVA , n = 3 , mean + SEM ) . GFP based reporter assay showing the efficiency of HR repair pathway under indicated siRNA - depleted conditions in HEK293 cells . Graph represents the quantifications from three independent experiments ( ns P > 0 . 05 , * * P < 0 . 01 ; one - way ANOVA , n = 3 , mean + SEM ) . CFA showing the IR sensitivity of 53BP1 - proficient and deficient MCF7 cells under indicated siRNA - depleted conditions . Data was collected from two individual experiments with triplicates . ( ns P > 0 . 05 ; two - way ANOVA , n = 2 , mean ± SEM ) . CFA showing the IR sensitivity of BRCA2 - proficient and deficient DLD1 cells under indicated siRNA - depleted conditions . Data was collected from two individual experiments with triplicates . ( * * P < 0 . 01 , * * * P < 0 . 001 ; two - way ANOVA , n = 2 , mean ± SEM ) . Representative IF - images showing Rad51 foci in S - phase ( Edu positive ) U2OS nucleus after 5h of IR ( 2Gy ) exposure . ( Scale bar ; 10µm ) . Graph represents the quantification of ( E ) showing average number of Rad51 foci per S - phase ( Edu positive ) nucleus . ( ns P > 0 . 05 , * * * * P < 0 . 0001 ; unpaired t - test , n = 2 , mean + SEM , at least 50 nuclei per condition and experiment ) . CFA showing the rescue of cell survival in IR treated , ATX3 - depleted ( siRNA ) , HeLa cells , after co - depletion ( siRNA ) of RNF8 . ( ns P > 0 . 05 , * P < 0 . 05 , * * * * P < 0 . 0001 ; two - way ANOVA , n = 3 , mean ± SEM ) .

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 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 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 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 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

Figure 8GFP based reporter assay showing the efficiency of NHEJ repair pathway under indicated siRNA-depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments (*P<0.05, **P<0.01, ***P<0.001; one-way ANOVA, n=3, mean +SEM).GFP based reporter assay showing the efficiency of HR repair pathway under indicated siRNA-depleted conditions in HEK293 cells. Graph represents the quantifications from three independent experiments (ns P>0.05, **P<0.01; one-way ANOVA, n=3, mean +SEM).CFA showing the IR sensitivity of 53BP1-proficient and deficient MCF7 cells under indicated siRNA-depleted conditions. Data was collected from two individual experiments with triplicates. (ns P>0.05; two-way ANOVA, n=2, mean ±SEM).CFA showing the IR sensitivity of BRCA2-proficient and deficient DLD1 cells under indicated siRNA-depleted conditions. Data was collected from two individual experiments with triplicates. (**P<0.01, ***P<0.001; two-way ANOVA, n=2, mean ±SEM).Representative IF-images showing Rad51 foci in S-phase (Edu positive) U2OS nucleus after 5h of IR (2Gy) exposure. (Scale bar; 10µm).   Graph represents the quantification of (E) showing average number of Rad51 foci per S-phase (Edu positive) nucleus. (ns P>0.05, ****P<0.0001; unpaired t-test, n=2, mean +SEM, at least 50 nuclei per condition and experiment).   CFA showing the rescue of cell survival in IR treated, ATX3-depleted (siRNA), HeLa cells, after co-depletion (siRNA) of RNF8. (ns P>0.05, *P<0.05, ****P<0.0001; two-way ANOVA, n=3, mean ±SEM).


Figure 1 ( A ) RT - PCR analysis for temporal expression of sinhcaf mRNA during embryogenesis and early larval developmental stages . NC indicates the no template control . ( B ) Time - course expression of sinhcaf revealed by whole - mount in situ hybridization . Scale bars , 100 μ ( C ) RT - PCR analysis for spatial expression of sinhcaf mRNA in adult tissues . ( E ) Relative mRNA level of sinhcaf in bud stage WT and MZsinhcaf - / - mutant embryos as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . ( F ) Fluorescence microscopy of 10hpf Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos . Scale bar , 400 μm . ( G ) Relative mRNA level of sinhcaf in 10hpf or 24hpf WT and Tg ( CMV : EGFP ; CMV : sinhcaf ) embryos as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using 2 - way ANOVA followed by Holm - Sidak post - hoc test . ( H ) Percentage of male zebrafish in WT ( n = 274 ) , MZsinhcaf - / - ( n = 166 ) , Tg ( CMV : EGFP ; CMV : sinhcaf ) ( n = 158 ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) ( n = 135 ) adult zebrafish . Data shown are mean ± SEM ( n = the number of fish analyzed ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 .

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

Figure 1(A) RT-PCR analysis for temporal expression of sinhcaf mRNA during embryogenesis and early larval developmental stages. NC indicates the no template control.(B) Time-course expression of sinhcaf revealed by whole-mount in situ hybridization. Scale bars, 100 μ(C) RT-PCR analysis for spatial expression of sinhcaf mRNA in adult tissues.(E) Relative mRNA level of sinhcaf in bud stage WT and MZsinhcaf -/- mutant embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test.(F) Fluorescence microscopy of 10hpf Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos. Scale bar, 400 μm.(G) Relative mRNA level of sinhcaf in 10hpf or 24hpf WT and Tg(CMV:EGFP;CMV:sinhcaf) embryos as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by Holm-Sidak post-hoc test.(H) Percentage of male zebrafish in WT (n = 274), MZsinhcaf -/- (n = 166), Tg(CMV:EGFP;CMV:sinhcaf) (n = 158) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) (n = 135) adult zebrafish. Data shown are mean ± SEM (n = the number of fish analyzed). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05.


Figure 2 ( A and B ) Whole - mount in situ hybridization was performed in WT , MZsinhcaf - / - , Tg ( CMV : EGFP ; CMV : sinhcaf ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos with PGCs markers nanos3 ( A ) and vasa ( B ) . Scale bars , 100 μm . ( C ) Quantitative analysis of PGCs in bud stage WT , MZsinhcaf - / - , Tg ( CMV : EGFP ; CMV : sinhcaf ) and MZsinhcaf - / - ; Tg ( CMV : EGFP ; CMV : sinhcaf ) zebrafish embryos . Data shown are mean ± SEM ( n = 10 , the number of embryos analyzed ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 . ( D ) Maximal intensity projection of a confocal z - stack of 24hpf Tg ( piwil1 : EGFP ) and MZsinhcaf - / - ; Tg ( piwil1 : EGFP ) zebrafish embryos , dorsal views . Arrowheads point to PGCs . Scale bars , 100 μm . ( E ) Quantitative analysis of PGCs in 24hpf Tg ( piwil1 : EGFP ) and MZsinhcaf - / - ; Tg ( piwil1 : EGFP ) zebrafish embryos . Data shown are mean ± SEM ( n = 13 , the number of embryos analyzed ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test .

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

Figure 2(A and B) Whole-mount in situ hybridization was performed in WT, MZsinhcaf -/-, Tg(CMV:EGFP;CMV:sinhcaf) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos with PGCs markers nanos3 (A) and vasa (B). Scale bars, 100 μm. (C) Quantitative analysis of PGCs in bud stage WT, MZsinhcaf -/-, Tg(CMV:EGFP;CMV:sinhcaf) and MZsinhcaf -/-;Tg(CMV:EGFP;CMV:sinhcaf) zebrafish embryos. Data shown are mean ± SEM (n = 10, the number of embryos analyzed). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05.(D) Maximal intensity projection of a confocal z-stack of 24hpf Tg(piwil1:EGFP) and MZsinhcaf -/-; Tg(piwil1:EGFP) zebrafish embryos, dorsal views. Arrowheads point to PGCs. Scale bars, 100 μm. (E) Quantitative analysis of PGCs in 24hpf Tg(piwil1:EGFP) and MZsinhcaf -/-; Tg(piwil1:EGFP)zebrafish embryos. Data shown are mean ± SEM (n = 13, the number of embryos analyzed). Statistical analysis was performed using unpaired two-tailed Student's t-test.


Figure 3 ( A ) Whole - mount in situ hybridization was performed in 4 - cell stage WT , MZsinhcaf - / - zebrafish embryos with germ plasm marker vasa . Red arrowheads point to cleavage furrows in the 4 - cell embryos . Scale bars , 100 μm ( B ) Relative mRNA levels of germ plasm genes vasa , dnd , nanos3 , dazl and buc in WT and MZsinhcaf - / - mutant eggs , respectively , as measured by RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using 2 - way ANOVA followed by the Holm - Sidak post - hoc test ; NS , no significant difference . ( C ) Maximal intensity projection of a confocal z - stack of 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP mRNA . White arrowheads point to cleavage furrows of 4 - cell embryos . Scale bars , 100 μm . ( D ) Relative GFP - Buc density at the cleavage furrow of 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP mRNA . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data shown are mean ± SEM ( n = 12 , the number of embryos analyzed ) . ( E ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . ( F ) Western blots for GFP - Buc expression in 4 - cell stage WT and MZsinhcaf - / - zebrafish embryos injected with GFP - Buc and RFP

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

Figure 3(A) Whole-mount in situ hybridization was performed in 4-cell stage WT, MZsinhcaf -/- zebrafish embryos with germ plasm marker vasa. Red arrowheads point to cleavage furrows in the 4-cell embryos. Scale bars, 100 μm(B) Relative mRNA levels of germ plasm genes vasa, dnd, nanos3, dazl and buc in WT and MZsinhcaf -/- mutant eggs, respectively, as measured by RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using 2-way ANOVA followed by the Holm-Sidak post-hoc test; NS, no significant difference.(C) Maximal intensity projection of a confocal z-stack of 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA. White arrowheads point to cleavage furrows of 4-cell embryos. Scale bars, 100 μm.(D) Relative GFP-Buc density at the cleavage furrow of 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA. Statistical analysis was performed using unpaired two-tailed Student's t-test. Data shown are mean ± SEM (n = 12, the number of embryos analyzed).(E) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test.(F) Western blots for GFP-Buc expression in 4-cell stage WT and MZsinhcaf -/- zebrafish embryos injected with GFP-Buc and RFP mRNA


Figure 4 ( A , B and C ) Co - immunoprecipitation of zebrafish Sinhcaf - GFP with HA - Hdac1 ( A ) , HA - Sin3a ( B ) or HA - Sap18 ( C ) in HEK293Tcells transfected with both Sinhcaf - GFP and HA - Hdac1 ( A ) , HA - Sin3a ( B ) or HA - Sap18 ( C ) plasmids . ( D ) Relative mRNA level of sinhcaf in zebrafish PG , PV , EV , MV and FG follicles was measured using RT - qPCR . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) . ( E and F ) Western blots for acetyl - histone H3 ( K9 ) ( Ace - H3 - K9 ) , histone H3 in WT ( E ) or MZsinhcaf - / - ( F ) PG , PV , EV , MV and FG follicles . α - tubulin was used as a loading control . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) . ( G and H ) Statistical analysis of the relative levels of Ace - H3 - K9 in WT ( G ) or MZsinhcaf - / - ( H ) PG , PV , EV , MV and FG follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . The different letters ( a , b , c ) indicate a significant difference between the means , p < 0 . 05 . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ; PG , primary growth ( stage I ) ; PV , previtellogenic stage ( stage II ) . ( I , J and K ) Western blots for Ace - H3 - K9 and H3 in WT and MZsinhcaf - / - EV ( I ) , MV ( J ) and FG ( K ) follicles . α - tubulin was used as a loading control . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ( L ) Western blots for Ace - H4 - K5 , Ace - H3 - K18 and H3 in WT and MZsinhcaf - / - FG follicles . α - tubulin was used as a loading control . Data information : FG , full - grown stage ( late stage III ) ( M , N and O ) Statistical analysis of the relative levels of Ace - H3 - K9I n WT and MZsinhcaf - / - EV ( M ) , MV ( N ) and FG ( O ) follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data information : EV , early vitellogenic stage ( early stage III ) ; FG , full - grown stage ( late stage III ) ; MV , mid vitellogenic stage ( mid stage III ) ( P and Q ) Statistical analysis of the relative levels of Ace - H3 - K18 ( P ) and Ace - H4 - K5 ( Q ) in WT and MZsinhcaf - / - FG follicles . Data shown are mean ± SEM ( n = 3 , biological replicates ) . Statistical analysis was performed using unpaired two - tailed Student ' s t - test . Data information : ; FG , full - grown stage ( late stage III )

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

Figure 4(A, B and C) Co-immunoprecipitation of zebrafish Sinhcaf-GFP with HA-Hdac1 (A), HA-Sin3a (B) or HA-Sap18 (C) in HEK293Tcells transfected with both Sinhcaf-GFP and HA-Hdac1 (A), HA-Sin3a (B) or HA-Sap18 (C) plasmids.(D) Relative mRNA level of sinhcaf in zebrafish PG, PV, EV, MV and FG follicles was measured using RT-qPCR. Data shown are mean ± SEM (n = 3, biological replicates). Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).(E and F) Western blots for acetyl-histone H3 (K9) (Ace-H3-K9), histone H3 in WT (E) or MZsinhcaf -/- (F) PG, PV, EV, MV and FG follicles. α-tubulin was used as a loading control. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).(G and H) Statistical analysis of the relative levels of Ace-H3-K9 in WT (G) or MZsinhcaf -/- (H) PG, PV, EV, MV and FG follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. The different letters (a, b, c) indicate a significant difference between the means, p<0.05. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III); PG, primary growth (stage I); PV, previtellogenic stage (stage II).(I, J and K) Western blots for Ace-H3-K9 and H3 in WT and MZsinhcaf -/- EV (I), MV (J) and FG (K) follicles. α-tubulin was used as a loading control. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III)(L) Western blots for Ace-H4-K5, Ace-H3-K18 and H3 in WT and MZsinhcaf -/- FG follicles. α-tubulin was used as a loading control. Data information: FG, full-grown stage (late stage III)(M, N and O) Statistical analysis of the relative levels of Ace-H3-K9I n WT and MZsinhcaf -/- EV (M), MV (N) and FG (O) follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. Data information: EV, early vitellogenic stage (early stage III); FG, full-grown stage (late stage III); MV, mid vitellogenic stage (mid stage III)(P and Q) Statistical analysis of the relative levels of Ace-H3-K18 (P) and Ace-H4-K5 (Q) in WT and MZsinhcaf -/- FG follicles. Data shown are mean ± SEM (n = 3, biological replicates). Statistical analysis was performed using unpaired two-tailed Student's t-test. Data information: ; FG, full-grown stage (late stage III)


Figure 7 ( A ) Maximal intensity projection of a confocal z - stack of 24hpf Tg ( piwil : egfp ) , Tg ( piwil : egfp ) injected with kif26ab MO and Tg ( piwil : egfp ) injected with control MO embryos . ( B ) Quantitative analysis of PGCs in 24hpf Tg ( piwil : egfp ) ( n = 11 ) , Tg ( piwil : egfp ) injected with kif26ab MO ( n = 12 ) and Tg ( piwil : egfp ) injected with control MO ( n = 13 ) embryos . Data shown are mean ± SEM ( n = the number of embryos analyzed ) . ( C ) Maximal intensity projection of a confocal z - stack of 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . White arrowheads point to cleavage furrows of 4 - cell embryos . ( D ) Relative GFP - Buc density at the cleavage furrow of 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA ( n = 11 ) , GFP - Buc mRNA + RFP mRNA + Control MO ( n = 11 ) and GFP - Buc mRNA + RFP mRNA + kif26ab MO ( n = 10 ) . Data shown are mean ± SEM ( n = the number of embryos analyzed ) . ( E ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . ( F ) Western blots for GFP - Buc expression in 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . Data information : Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . Scale bars , 100 μm . ( G ) Maximal intensity projection of a confocal z - stack of MZsinhcaf - / - or WT 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA or GFP - Buc mRNA + RFP mRNA + kif26ab mRNA . White arrowheads point to cleavage furrows of 4 - cell embryos . ( H ) Relative GFP - Buc density at the cleavage furrow of MZsinhcaf - / - ( n = 12 ) or WT ( n = 13 ) 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA or GFP - Buc mRNA + RFP mRNA + kif26ab mRNA . Data shown are mean ± SEM ( n = the number of embryos analyzed ) . ( I ) Statistical analysis of the relative levels of GFP - Buc / a - Tubulin . Data shown are mean ± SEM ( n = 3 , biological replicates ) . ( J ) Western blots for GFP - Buc expression in MZsinhcaf - / - or WT 4 - cell stage embryos injected with GFP - Buc mRNA + RFP mRNA , GFP - Buc mRNA + RFP mRNA + Control MO and GFP - Buc mRNA + RFP mRNA + kif26ab MO . Data information : Statistical analysis was performed using one - way ANOVA followed by Tukey ' s post - hoc test . Scale bars , 100 μm .

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

Figure 7(A) Maximal intensity projection of a confocal z-stack of 24hpf Tg(piwil:egfp), Tg(piwil:egfp) injected with kif26ab MO and Tg(piwil:egfp) injected with control MO embryos. (B) Quantitative analysis of PGCs in 24hpf Tg(piwil:egfp) (n = 11), Tg(piwil:egfp) injected with kif26ab MO (n = 12) and Tg(piwil:egfp) injected with control MO (n = 13) embryos. Data shown are mean ± SEM (n = the number of embryos analyzed).(C) Maximal intensity projection of a confocal z-stack of 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. White arrowheads point to cleavage furrows of 4-cell embryos.(D) Relative GFP-Buc density at the cleavage furrow of 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA (n = 11), GFP-Buc mRNA + RFP mRNA + Control MO (n = 11) and GFP-Buc mRNA + RFP mRNA + kif26ab MO (n = 10). Data shown are mean ± SEM (n = the number of embryos analyzed).(E) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). (F) Western blots for GFP-Buc expression in 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. Data information: Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Scale bars, 100 μm.(G) Maximal intensity projection of a confocal z-stack of MZsinhcaf -/- or WT 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA or GFP-Buc mRNA + RFP mRNA + kif26ab mRNA. White arrowheads point to cleavage furrows of 4-cell embryos.(H) Relative GFP-Buc density at the cleavage furrow of MZsinhcaf -/- (n = 12) or WT (n = 13) 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA or GFP-Buc mRNA + RFP mRNA + kif26ab mRNA. Data shown are mean ± SEM (n = the number of embryos analyzed).(I) Statistical analysis of the relative levels of GFP-Buc/a-Tubulin. Data shown are mean ± SEM (n = 3, biological replicates). (J) Western blots for GFP-Buc expression in MZsinhcaf -/- or WT 4-cell stage embryos injected with GFP-Buc mRNA + RFP mRNA, GFP-Buc mRNA + RFP mRNA + Control MO and GFP-Buc mRNA + RFP mRNA + kif26ab MO. Data information: Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. Scale bars, 100 μm.


f1 ( a ) Confocal analysis of the subcellular localization of HA - PLD1 ( green ) and endogenous LC3 ( red ) in HeLa cells in normal medium and under nutrient deprivation conditions . Left panels : normal medium condition ( Nm ) ; middle panels : 1 h with nutrient - deprived medium ( 1 h St ) ; right panels : 2 h with nutrient - deprived medium ( 2 h St ) . Insets denote high magnification of the selected area . Scale bar , 5 μm . ( b ) Immuno - gold EM micrographs of HeLa cells transfected with GFP - PLD1 and subjected to 1 h of nutrient deprivation in the presence ( St + B ) or absence ( St ) of bafilomycin ( 50 nM ) . Insets denote high magnification of the selected area . Scale bar , 500 nm . Right panel : Histogram showing morphometric analysis of the relative distribution of GFP - PLD1 - positive gold particles on the outer ( arrows ) and inner ( arrowheads ) membranes of the autophagosomal - like structures . Values denote means ± s . e . m . ( n = 14 ) . * * P0 . 01 .

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

f1(a) Confocal analysis of the subcellular localization of HA-PLD1 (green) and endogenous LC3 (red) in HeLa cells in normal medium and under nutrient deprivation conditions. Left panels: normal medium condition (Nm); middle panels: 1 h with nutrient-deprived medium (1 h St); right panels: 2 h with nutrient-deprived medium (2 h St). Insets denote high magnification of the selected area. Scale bar, 5 μm.(b) Immuno-gold EM micrographs of HeLa cells transfected with GFP-PLD1 and subjected to 1 h of nutrient deprivation in the presence (St+B) or absence (St) of bafilomycin (50 nM). Insets denote high magnification of the selected area. Scale bar, 500 nm. Right panel: Histogram showing morphometric analysis of the relative distribution of GFP-PLD1-positive gold particles on the outer (arrows) and inner (arrowheads) membranes of the autophagosomal-like structures. Values denote means±s.e.m. (n=14). **P0.01.


f2Confocal analysis of the subcellular localization of HA - PLD1 and endogenous LC3 using various organelle markers in cells deprived of nutrients for 2 h . Organelle markers used were as follows ( from top to bottom panels ) : Lamp1 ( late endosomes / lysosomes ) , giantin ( Golgi - TGN ) , Vps35 ( retromer - positive endosomes ) , GFP - Rab5 ( early endosomes ) , GFP - UVRAG ( early and late endosomes ) . Insets show high magnification of the selected areas . The triple insets at the bottom of each column of pictures indicate high magnification of the selected areas for two channels in various combinations . Scale bar , 5 μm .

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f2Confocal analysis of the subcellular localization of HA-PLD1 and endogenous LC3 using various organelle markers in cells deprived of nutrients for 2 h. Organelle markers used were as follows (from top to bottom panels): Lamp1 (late endosomes/lysosomes), giantin (Golgi-TGN), Vps35 (retromer-positive endosomes), GFP-Rab5 (early endosomes), GFP-UVRAG (early and late endosomes). Insets show high magnification of the selected areas. The triple insets at the bottom of each column of pictures indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm.


f3 ( a ) Confocal analysis of HeLa cells subjected to nutrient deprivation for 2 h . Cells were transfected with HA - PLD1 and GFP - ATG14L ( upper panels ) or with HA - PLD1 alone ( lower panels ) . Upper panels : HA - PLD1 ( red ) , GFP - ATG14L ( green ) , endogenous LC3 ( blue ) . Lower panels : HA - PLD1 ( green ) , endogenous ATG16L ( red ) and LC3 ( blue ) . Insets show high magnification of the selected areas . The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations . Scale bar , 5 μm . ( b ) Confocal analysis showing lack of co - localization of HA - PLD1 ( green ) with the isolation membrane marker ATG16L ( blue ) in HeLa cells expressing the strawberry - ATG4 - C74A mutant ( red ) and subjected to 1 h of nutrient deprivation . Insets denote high magnification of the selected area . Scale bar , 5 μm . ( c ) EM analysis showing that GFP - PLD1 does not localize to the isolation membrane ( arrows ) in cells expressing the strawberry - ATG4 - C74A mutant . Immuno - gold EM micrographs of HeLa cells transfected with GFP - PLD1 and pStrawberry - ATG4 - C74A and subjected to 1 h of nutrient deprivation . Inset denotes high magnification of the selected area . Scale bar , 250 nm .

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 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

f3(a) Confocal analysis of HeLa cells subjected to nutrient deprivation for 2 h. Cells were transfected with HA-PLD1 and GFP-ATG14L (upper panels) or with HA-PLD1 alone (lower panels). Upper panels: HA-PLD1 (red), GFP-ATG14L (green), endogenous LC3 (blue). Lower panels: HA-PLD1 (green), endogenous ATG16L (red) and LC3 (blue). Insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations. Scale bar, 5 μm.(b) Confocal analysis showing lack of co-localization of HA-PLD1 (green) with the isolation membrane marker ATG16L (blue) in HeLa cells expressing the strawberry-ATG4-C74A mutant (red) and subjected to 1 h of nutrient deprivation. Insets denote high magnification of the selected area. Scale bar, 5 μm.(c) EM analysis showing that GFP-PLD1 does not localize to the isolation membrane (arrows) in cells expressing the strawberry-ATG4-C74A mutant. Immuno-gold EM micrographs of HeLa cells transfected with GFP-PLD1 and pStrawberry-ATG4-C74A and subjected to 1 h of nutrient deprivation. Inset denotes high magnification of the selected area. Scale bar, 250 nm.


f4 ( a ) Histogram showing relative phospholipid levels in CHO cells treated with 0 . 3 % ethanol for 30 min before cell harvesting . Total PLD activity is measured as phosphatidylethanol ( PEtOH ) levels under the following conditions : normal medium ( Nm ) , serum - free medium ( SFM ) , serum - free medium with 100 nM wortmannin ( SFM + W ) , nutrient deprivation ( 0 . 5 h St ; 1 . 5 h St ; 3 . 0 h St ) , 30 min of nutrient deprivation in presence of 100nM wortmannin ( 0 . 5h St + W ) . Other phospholipids were used as controls : phosphatidylserine ( PS ) , phosphatidylethanolamine ( PE ) , phosphatidylcholine ( PC ) . Values denote means ± s . e . m . ( n = 9 , except for SFM + W and 0 . 5 h St + W , where n = 3 and 5 , respectively ) . * * P0 . 01 ; * , P0 . 05 . ( b ) Western blot analysis of HA - PLD1 and endogenous LC3 levels in post - nuclear supernatant ( PNS , upper panel ) , as well as in cytosolic / soluble ( S ) and particulate ( P ) fractions prepared from PNS after high - speed centrifugation ( middle panel ) . GAPDH was used as loading control in PNS fractions . HeLa cells were transfected with expression vectors encoding HA - PLD1 for 24 h and subjected to 2 h of nutrient deprivation in the presence or absence of the PI3K inhibitor wortmannin ( 100 nM ) . Lower panel : quantitative analysis of HA - PLD1 immunoreactivity expressed as P / S ratio . Conditions were as follows : normal medium ( Nm ) ; normal medium with wortmannin ( Nm + W ) ; starvation condition ( St ) ; starvation condition + wortmannin ( St + W ) . Values denote means ± s . e . m . ( n = 3 ) . ( c ) Confocal analysis of the co - localization between HA - PLD1 and Lamp1 in HeLa cells subjected to nutrient deprivation for 2 h in the presence ( ST + W ) or absence ( St ) of wortmannin 100 nM . Insets show high magnification of the selected areas . Scale bar , 5 μm .

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

f4(a) Histogram showing relative phospholipid levels in CHO cells treated with 0.3% ethanol for 30 min before cell harvesting. Total PLD activity is measured as phosphatidylethanol (PEtOH) levels under the following conditions: normal medium (Nm), serum-free medium (SFM), serum-free medium with 100 nM wortmannin (SFM+W), nutrient deprivation (0.5 h St; 1.5 h St; 3.0 h St), 30 min of nutrient deprivation in presence of 100nM wortmannin (0.5h St+W). Other phospholipids were used as controls: phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylcholine (PC). Values denote means±s.e.m. (n=9, except for SFM+W and 0.5 h St+W, where n=3 and 5, respectively). **P0.01; *, P0.05.(b) Western blot analysis of HA-PLD1 and endogenous LC3 levels in post-nuclear supernatant (PNS, upper panel), as well as in cytosolic/soluble (S) and particulate (P) fractions prepared from PNS after high-speed centrifugation (middle panel). GAPDH was used as loading control in PNS fractions. HeLa cells were transfected with expression vectors encoding HA-PLD1 for 24 h and subjected to 2 h of nutrient deprivation in the presence or absence of the PI3K inhibitor wortmannin (100 nM). Lower panel: quantitative analysis of HA-PLD1 immunoreactivity expressed as P/S ratio. Conditions were as follows: normal medium (Nm); normal medium with wortmannin (Nm+W); starvation condition (St); starvation condition+wortmannin (St+W). Values denote means±s.e.m. (n=3).(c) Confocal analysis of the co-localization between HA-PLD1 and Lamp1 in HeLa cells subjected to nutrient deprivation for 2 h in the presence (ST+W) or absence (St) of wortmannin 100 nM. Insets show high magnification of the selected areas. Scale bar, 5 μm.


f5 ( a ) Western blot analysis showing the effects of siRNA - mediated Vps34 ( siVps34 ) downregulation on the expression levels of HA - PLD1 and Vps34 compared with control cells ( siCtrl ) . Cells were incubated in normal medium ( Nm ) or subjected to 2 h of nutrient deprivation in the presence of 50 nM bafilomycin ( St + B ) . HeLa cells were transfected for 48 h with siRNA directed to Vps34 ( siVps34 ) or a control sequence ( siCtrl ) and then transfected for 24 h with a plasmid encoding HA - PLD1 . ( b ) Confocal imaging showing the effects of Vps34 ( siVps34 ) downregulation on HA - PLD1 ( green ) , endogenous LC3 ( blue ) and Lamp1 ( red ) on 2 h of nutrient deprivation compared with control treatment ( siCtrl ) . Scale bar , 5 μm . ( c ) Confocal analysis showing the subcellular localization of HA - PLD1 mutated in its PX domain ( F120A / R179Q ) , so as to abolish phosphoinositide binding ( HA - PXmut ) . HeLa cells were transiently transfected with a plasmid encoding HA - PXmut and fixed after 2 h of nutrient deprivation . HA - PXmut ( green ) , endogenous LC3 ( blue ) and Lamp1 ( red ) . Scale bar , 10 μm . In b and c , insets show high magnification of the selected areas . The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f5(a) Western blot analysis showing the effects of siRNA-mediated Vps34 (siVps34) downregulation on the expression levels of HA-PLD1 and Vps34 compared with control cells (siCtrl). Cells were incubated in normal medium (Nm) or subjected to 2 h of nutrient deprivation in the presence of 50 nM bafilomycin (St+B). HeLa cells were transfected for 48 h with siRNA directed to Vps34 (siVps34) or a control sequence (siCtrl) and then transfected for 24 h with a plasmid encoding HA-PLD1.(b) Confocal imaging showing the effects of Vps34 (siVps34) downregulation on HA-PLD1 (green), endogenous LC3 (blue) and Lamp1 (red) on 2 h of nutrient deprivation compared with control treatment (siCtrl). Scale bar, 5 μm. (c) Confocal analysis showing the subcellular localization of HA-PLD1 mutated in its PX domain (F120A/R179Q), so as to abolish phosphoinositide binding (HA-PXmut). HeLa cells were transiently transfected with a plasmid encoding HA-PXmut and fixed after 2 h of nutrient deprivation. HA-PXmut (green), endogenous LC3 (blue) and Lamp1 (red). Scale bar, 10 μm. In b and c, insets show high magnification of the selected areas. The triple insets on the right indicate high magnification of the selected areas for two channels in various combinations.