Figure 5Phenotypes of embryos . Typical embryos , cropped from representative experimental groups in , are shown in ( B ) . Only GFP - positive embryos viable at 2 . 5 hpf were collected and imaged for analyses . Phenotypes of embryos . Their phenotypes served as criteria for quantifications in ( C ) . Only GFP - positive embryos viable at 2 . 5 hpf were collected and imaged for analyses . Total embryo numbers analyzed are listed over histograms . D . Hexanediol ( Hex ) treatment from 2 . 5 hpf blocked the blastula development . Representative images are presented for each group of embryos , with phenotypes indicated by insets of colored boxes . Total embryo numbers analyzed are listed over histograms .

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Figure 5Phenotypes of embryos. Typical embryos, cropped from representative experimental groups in , are shown in (B). Only GFP-positive embryos viable at 2.5 hpf were collected and imaged for analyses.Phenotypes of embryos. Their phenotypes served as criteria for quantifications in (C). Only GFP-positive embryos viable at 2.5 hpf were collected and imaged for analyses. Total embryo numbers analyzed are listed over histograms.D. Hexanediol (Hex) treatment from 2.5 hpf blocked the blastula development. Representative images are presented for each group of embryos, with phenotypes indicated by insets of colored boxes. Total embryo numbers analyzed are listed over histograms.


Figure 6C . Cumulative distribution showing fold changes ( FCs ) in transcript abundance in control morphants ( blue curves ) and maternal zRbm14 morphants ( red curves ) between the indicated stages . Transcript abundances were averaged from two independent experiments . D . Expression patterns of four clusters of representative transcripts , categorized from RNA - seq results of ctrl - MO embryos . The transcripts in the maternal - early and the zygotic - early clusters respectively displayed continual degradation or accumulation from 2 . 5 hpf , whereas those in the maternal - late and zygotic - late clusters respectively started degradation or accumulation from 4 hpf . E . Cumulative distribution showing FCs of transcript abundance of the indicated clusters between control and maternal zRbm14 morphants . F . Relative transcript levels of three typical maternal genes . FCs were calculated relatively to the levels in 2 . 5 - hpf ctrl - MO embryos . org , oogenesis - related ; trip10a , thyroid hormone receptor interactor 10a ; dnajc5ga , DnaJ ( Hsp40 ) homolog , subfamily C , member 5 gamma a . G . Whole - mount in situ RNA hybridization of org , trip10a , and dnajc5ga . A representative embryo is shown for each group of at least 26 embryos . EU incorporated into nascent transcripts were visualized through a click reaction . Hoechst 33342 was used to counterstain the nucleus . A representative maximum intensity - projected confocal micrograph , taken from the animal pole , is shown for each group of 10 embryos ( I ) .

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Figure 6C. Cumulative distribution showing fold changes (FCs) in transcript abundance in control morphants (blue curves) and maternal zRbm14 morphants (red curves) between the indicated stages. Transcript abundances were averaged from two independent experiments.D. Expression patterns of four clusters of representative transcripts, categorized from RNA-seq results of ctrl-MO embryos. The transcripts in the maternal-early and the zygotic-early clusters respectively displayed continual degradation or accumulation from 2.5 hpf, whereas those in the maternal-late and zygotic-late clusters respectively started degradation or accumulation from 4 hpf.E. Cumulative distribution showing FCs of transcript abundance of the indicated clusters between control and maternal zRbm14 morphants.F. Relative transcript levels of three typical maternal genes. FCs were calculated relatively to the levels in 2.5-hpf ctrl-MO embryos. org, oogenesis-related; trip10a, thyroid hormone receptor interactor 10a; dnajc5ga, DnaJ (Hsp40) homolog, subfamily C, member 5 gamma a.G. Whole-mount in situ RNA hybridization of org, trip10a, and dnajc5ga. A representative embryo is shown for each group of at least 26 embryos.EU incorporated into nascent transcripts were visualized through a click reaction. Hoechst 33342 was used to counterstain the nucleus. A representative maximum intensity-projected confocal micrograph, taken from the animal pole, is shown for each group of 10 embryos (I).


Figure 7Depletion of zRbm14 resulted in accumulation of poly ( A ) - containing maternal mRNAs . Total RNAs purified from the indicated zebrafish embryos were subjected to poly ( A ) length assays . One set of representative PCR results for org and trip10a ( B ) As the PCR products of the poly ( A ) - transcripts mainly emerged as a single band , only intensities of this major band were measured . Depletion of zRbm14 resulted in accumulation of poly ( A ) - containing maternal mRNAs . Total RNAs purified from the indicated zebrafish embryos were subjected to poly ( A ) length assays . quantification results from three independent experiments ( C ) are presented . As the PCR products of the poly ( A ) - transcripts mainly emerged as a single band , only intensities of this major band were measured . D . zParn associated with zRbm14b . GFP - zRbm14b was co - expressed respectively with the indicated RFP fusion proteins in HEK293T cells . Co - immunoprecipitations ( co - IP ) were performed using anti - GFP beads . Luciferase ( Luc ) - RFP served as a negative control . E , F . zParn markedly promoted LLPS of zRbm14b by interplaying with zIDR . Purified His - tagged RFP or zParn - RFP was mixed with His - tagged GFP or GFP - zRbm14b or mutants ( E ) . Parn - RFP was mixed with decreasing concentrations of GFP - zRbm14b ( F ) . G , H . zParn was highly expressed from 4 - cell to 32 - cell stages and enriched in zRbm14 condensates . Immunoblotting ( G ) and immunostaining ( H ) were performed using a rabbit anti - zebrafish Parn antibody . Lysates from 5 embryos were loaded in each lane in ( G ) . α - tubulin served as internal controls . Representative single optical sections are presented in ( H ) . Chromatin DNA was stained with DAPI . Arrows point to typical regions abundant in condensates . J . zRbm14b and zParn efficiently co - phase separated in deadenylation experiments . The addition of SDS disrupted the condensates to terminate the deadenylation reaction . Co - phase separation with zRbm14b markedly enhanced the deadenylation activity of zParn . RNAs extracted from the indicated samples were subjected to immuno - northern blotting using an antibody against m6A ( K ) . were quantified from five sets of independent results . Co - phase separation with zRbm14b markedly enhanced the deadenylation activity of zParn . Relatively proportions of Cap - 10m6A - A0 in total RNAs ( L ) were quantified from five sets of independent results .

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

Figure 7Depletion of zRbm14 resulted in accumulation of poly(A)-containing maternal mRNAs. Total RNAs purified from the indicated zebrafish embryos were subjected to poly(A) length assays. One set of representative PCR results for org and trip10a (B) As the PCR products of the poly(A)-transcripts mainly emerged as a single band, only intensities of this major band were measured.Depletion of zRbm14 resulted in accumulation of poly(A)-containing maternal mRNAs. Total RNAs purified from the indicated zebrafish embryos were subjected to poly(A) length assays. quantification results from three independent experiments (C) are presented. As the PCR products of the poly(A)-transcripts mainly emerged as a single band, only intensities of this major band were measured.D. zParn associated with zRbm14b. GFP-zRbm14b was co-expressed respectively with the indicated RFP fusion proteins in HEK293T cells. Co-immunoprecipitations (co-IP) were performed using anti-GFP beads. Luciferase (Luc)-RFP served as a negative control.E, F. zParn markedly promoted LLPS of zRbm14b by interplaying with zIDR. Purified His-tagged RFP or zParn-RFP was mixed with His-tagged GFP or GFP-zRbm14b or mutants (E). Parn-RFP was mixed with decreasing concentrations of GFP-zRbm14b (F).G, H. zParn was highly expressed from 4-cell to 32-cell stages and enriched in zRbm14 condensates. Immunoblotting (G) and immunostaining (H) were performed using a rabbit anti-zebrafish Parn antibody. Lysates from 5 embryos were loaded in each lane in (G). α-tubulin served as internal controls. Representative single optical sections are presented in (H). Chromatin DNA was stained with DAPI. Arrows point to typical regions abundant in condensates.J. zRbm14b and zParn efficiently co-phase separated in deadenylation experiments. The addition of SDS disrupted the condensates to terminate the deadenylation reaction.Co-phase separation with zRbm14b markedly enhanced the deadenylation activity of zParn. RNAs extracted from the indicated samples were subjected to immuno-northern blotting using an antibody against m6A (K). were quantified from five sets of independent results.Co-phase separation with zRbm14b markedly enhanced the deadenylation activity of zParn. Relatively proportions of Cap-10m6A-A0 in total RNAs (L) were quantified from five sets of independent results.


Figure 8B . Mouse Rbm14 formed cytoplasmic puncta in blastomeres ( 1 - cell to 8 - cell ) . Maximum intensity - projected confocal immunofluorescent micrographs are presented . Framed regions are magnified to show details of cytoplasmic Rbm14 puncta . Arrows point to Rbm14 condensates accumulated at spindle poles . D . Relative transcript levels of Rbm14 in the indicated E4 . 5 blastocysts based on RNA deep Sequencing results , normalized to those of Gapdh . Six blastocysts were considered as Rbm14 - / - embryos because they contained only trace amount of Rbm14 transcript reminiscent of maternal mRNA . F . Heat map of differentially expressed genes . G . Heat map of representative lineage - specific transcripts . TE , trophectoderm ; EPI , epiblast ; PrE , primitive endoderm . Severe down - regulation of Gata6 and Nanog in Rbm14 - depleted mouse blastocysts . Representative immunofluorescent images ( H ) were from single option sections . DAPI stained nuclear DNA . As Gata6 , Nanog , and Rbm14 localize to the nucleus in blastocystsSevere down - regulation of Gata6 and Nanog in Rbm14 - depleted mouse blastocysts . As Gata6 , Nanog , and Rbm14 localize to the nucleus in blastocysts , their relative intensity in a cell ( I ) was quantified as their nuclear immunofluorescent intensity normalized with fluorescent intensity of the nuclear DNA . Five brightest cells were quantified for each embryo .

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Figure 8B. Mouse Rbm14 formed cytoplasmic puncta in blastomeres (1-cell to 8-cell). Maximum intensity-projected confocal immunofluorescent micrographs are presented. Framed regions are magnified to show details of cytoplasmic Rbm14 puncta. Arrows point to Rbm14 condensates accumulated at spindle poles.D. Relative transcript levels of Rbm14 in the indicated E4.5 blastocysts based on RNA deep Sequencing results, normalized to those of Gapdh. Six blastocysts were considered as Rbm14-/- embryos because they contained only trace amount of Rbm14 transcript reminiscent of maternal mRNA.F. Heat map of differentially expressed genes.G. Heat map of representative lineage-specific transcripts. TE, trophectoderm; EPI, epiblast; PrE, primitive endoderm.Severe down-regulation of Gata6 and Nanog in Rbm14-depleted mouse blastocysts. Representative immunofluorescent images (H) were from single option sections. DAPI stained nuclear DNA. As Gata6, Nanog, and Rbm14 localize to the nucleus in blastocystsSevere down-regulation of Gata6 and Nanog in Rbm14-depleted mouse blastocysts. As Gata6, Nanog, and Rbm14 localize to the nucleus in blastocysts, their relative intensity in a cell (I) was quantified as their nuclear immunofluorescent intensity normalized with fluorescent intensity of the nuclear DNA. Five brightest cells were quantified for each embryo.


Figure 1A - C Confocal images of primary hepatocytes grown under indicated conditions and stained with anti - CLUH antibody and Hadha ( A ) , Pcx ( B ) and Actb ( C ) mRNA in situ hybridization . Right panels show 5x magnified boxed areas . Scale bar , 10 μm . D - F Manders ' colocalization coefficient between Hadha ( D ) Pcx ( E ) and Actb ( F ) mRNA molecules and CLUH signal ( n ≥ 50 cells isolated from 3 - 6 mice ) . Data information : In ( D - F ) , data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ( Student ' s t - test ) . G , H Confocal images of primary hepatocytes grown in HBSS and stained with anti - CLUH and anti - TIA - 1 ( G ) or anti - G3BP1 ( H ) antibodies . Right panels show 5x magnified boxed areas . Scale bar , 10 μm .

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Figure 1A-C Confocal images of primary hepatocytes grown under indicated conditions and stained with anti-CLUH antibody and Hadha (A), Pcx (B) and Actb (C) mRNA in situ hybridization. Right panels show 5x magnified boxed areas. Scale bar, 10 μm. D-F Manders' colocalization coefficient between Hadha (D) Pcx (E) and Actb (F) mRNA molecules and CLUH signal (n ≥ 50 cells isolated from 3-6 mice). Data information: In (D-F), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001 (Student's t-test).G, H Confocal images of primary hepatocytes grown in HBSS and stained with anti-CLUH and anti-TIA-1 (G) or anti-G3BP1 (H) antibodies. Right panels show 5x magnified boxed areas. Scale bar, 10 μm.


Figure 2A Confocal images of primary hepatocytes grown under indicated conditions and treatments after ribopuromycylation assay stained with anti - puromycin antibody . Scale bar , 10 μm . B Quantification of fluorescence intensity per cell of experiment shown in ( A ) . AU , arbitrary units . ( n = 90 - 110 cells per treatment isolated from 4 mice ) . Data information data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . C Quantification of the number of puromycin granules under indicated conditions ( n ≥ 50 cells isolated from 4 mice ) . Data information : data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ; * * , P ≤ 0 . 01 ( Student ' s t - test ) . D Confocal images of primary hepatocytes stained with anti - puromycin and anti - CLUH antibodies . The cells from which the enlarged areas ( 400 μm2 ) have been magnified are shown in Appendix Figure S1B for each individual channel . Scale bar , 4 μm . Cells analyzed were isolated from 4 different mice with similar results . Arrows point to colocalizing particles . E Manders ' colocalization coefficient between puromycin and CLUH from experiment shown in ( D ) ( n ≥ 50 cells isolated from 4 mice ) . Data information : data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . ( E ) * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . F Quantification of puromycin granules containing CLUH signal ( n ≥ 80 cells isolated from 4 mice ) . Data information : data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ; * * , P ≤ 0 . 01 ( Student ' s t - test ) .

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Figure 2A Confocal images of primary hepatocytes grown under indicated conditions and treatments after ribopuromycylation assay stained with anti-puromycin antibody. Scale bar, 10 μm. B Quantification of fluorescence intensity per cell of experiment shown in (A). AU, arbitrary units. (n= 90-110 cells per treatment isolated from 4 mice). Data information data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values.C Quantification of the number of puromycin granules under indicated conditions (n ≥ 50 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001; **, P≤0.01 (Student's t-test).D Confocal images of primary hepatocytes stained with anti-puromycin and anti-CLUH antibodies. The cells from which the enlarged areas (400 μm2) have been magnified are shown in Appendix Figure S1B for each individual channel. Scale bar, 4 μm. Cells analyzed were isolated from 4 different mice with similar results. Arrows point to colocalizing particles.E Manders' colocalization coefficient between puromycin and CLUH from experiment shown in (D) (n ≥ 50 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (E) ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).F Quantification of puromycin granules containing CLUH signal (n ≥ 80 cells isolated from 4 mice). Data information: data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001; **, P≤0.01 (Student's t-test).


Figure 3A - C Confocal images of primary hepatocytes after ribopuromycylation experiment combined with mRNA in situ hybridization for ( A ) Hadha , ( B ) Pcx and ( C ) Hmgcs2 . Scale bar , 4 µm . D - F Fluorescence profile of 100 - pixel line from the merged channel shown in ( A - C ) . The cells from which the enlarged areas ( 400 μm2 ) have been magnified are shown in Appendix Figure S2 for each individual channel . Cells analyzed were isolated from 6 different mice with similar results .

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Figure 3A-C Confocal images of primary hepatocytes after ribopuromycylation experiment combined with mRNA in situ hybridization for (A) Hadha, (B) Pcx and (C) Hmgcs2. Scale bar, 4 µm. D-F Fluorescence profile of 100-pixel line from the merged channel shown in (A-C). The cells from which the enlarged areas (400 μm2) have been magnified are shown in Appendix Figure S2 for each individual channel. Cells analyzed were isolated from 6 different mice with similar results.


Figure 4A Representative western blot of HeLa cells downregulated for G3BPs and overexpressing untagged CLUH . Asterisks indicates signal of previous incubation with anti - G3BP1 antibody . Pan - actin was used as loading control . B Confocal images of anti - CLUH staining in HeLa cells downregulated for G3BPs and overexpressing untagged CLUH . Insets show 2 . 6x enlarged area . Scale bar , 10 µm . C Quantification of cells with CLUH - granules from experiments shown in ( B ) ( n = 3 independent experiments , > 50 cells per experiment per condition ) . Data information data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . D Life imaging of WT and CLUH KO HeLa cells transfected with G3BP1 - GFP plasmid and incubated in HBSS medium with or without CHX . Cells were recorded for a maximum of 2 h . Insets show 2 . 5x enlarged areas . Scale bar , 10 µm . E Total number of cells analyzed by life imaging for the indicated experiments shown in ( D ) . " Positive " relates to a cell which formed G3BP1 granules at the end of the recording . F Confocal images of anti - G3BP1 staining in primary hepatocytes derived from Li - CluhWT and Li - CluhKO mice grown in indicated media . Arrows point to G3BP1 - granules . Scale bar , 10 µm . G Quantification of percentage of cells with G3BP1 - positive granules of experiment shown in F . Cells were analyzed in a blind fashion ( n = 3 mice per genotype ; number of cells in total : WT Basal , 449 ; KO Basal , 303 ; WT HBSS , 146 ; KO HBSS , 161 ) . Data information : , data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . H Western blot of primary WT and KO hepatocytes stained with indicated antibodies . Asterisks indicates unspecific signal . Pan - actin was used as loading control . I Quantification of western blot ( n = 3 per genotype per condition ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . J mRNA levels of G3bp1 in primary hepatocytes grown under indicated conditions . ( n = 4 per genotype per condition ) . Data information data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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Figure 4A Representative western blot of HeLa cells downregulated for G3BPs and overexpressing untagged CLUH. Asterisks indicates signal of previous incubation with anti-G3BP1 antibody. Pan-actin was used as loading control.B Confocal images of anti-CLUH staining in HeLa cells downregulated for G3BPs and overexpressing untagged CLUH. Insets show 2.6x enlarged area. Scale bar, 10 µm. C Quantification of cells with CLUH-granules from experiments shown in (B) (n=3 independent experiments, >50 cells per experiment per condition). Data information data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test). D Life imaging of WT and CLUH KO HeLa cells transfected with G3BP1-GFP plasmid and incubated in HBSS medium with or without CHX. Cells were recorded for a maximum of 2 h. Insets show 2.5x enlarged areas. Scale bar, 10 µm. E Total number of cells analyzed by life imaging for the indicated experiments shown in (D). "Positive" relates to a cell which formed G3BP1 granules at the end of the recording. F Confocal images of anti-G3BP1 staining in primary hepatocytes derived from Li-CluhWT and Li-CluhKO mice grown in indicated media. Arrows point to G3BP1-granules. Scale bar, 10 µm. G Quantification of percentage of cells with G3BP1-positive granules of experiment shown in F. Cells were analyzed in a blind fashion (n=3 mice per genotype; number of cells in total: WT Basal, 449; KO Basal, 303; WT HBSS, 146; KO HBSS, 161). Data information: , data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test). H Western blot of primary WT and KO hepatocytes stained with indicated antibodies. Asterisks indicates unspecific signal. Pan-actin was used as loading control.I Quantification of western blot (n=3 per genotype per condition). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test). J mRNA levels of G3bp1 in primary hepatocytes grown under indicated conditions. (n=4 per genotype per condition). Data information data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


Figure 5A , B Volcano plots showing significantly changed proteins in KO hepatocytes relative to WT in basal ( A ) and HBSS ( B ) conditions . F Correlation of protein fold changes in KO in respect to WT hepatocytes under basal condition and upon HBSS ( 2 h ) . Only genes previously found in CLUH RIP experiments in HeLa cells ( Gao et al . , 2014 ) are plotted . Inset shows magnification of upper part of the graph .

O B-PANEL_START O O 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

Figure 5A, B Volcano plots showing significantly changed proteins in KO hepatocytes relative to WT in basal (A) and HBSS (B) conditions.F Correlation of protein fold changes in KO in respect to WT hepatocytes under basal condition and upon HBSS (2 h). Only genes previously found in CLUH RIP experiments in HeLa cells (Gao et al., 2014) are plotted. Inset shows magnification of upper part of the graph.


Figure 6A Representative western blot of primary hepatocytes starved in HBSS for the indicated time points and probed with indicated antibodies . Pan - actin was used as loading control . B , C Quantification of western blots as shown in ( A ) . Antibody signal was normalized to pan - actin signal and signal of phospho - protein was normalized to signal of total protein ( n = 4 independent experiments ) . Data information : In ( B , C ) , data shows the mean ± SEM . * * , P ≤ 0 . 01 for genotype and * * * , P ≤ 0 . 001 for time ( Two - way ANOVA ) . Error bars show minimum and maximum values . D Representative western blot of total protein extracts from livers of starved Li - CluhWT and Li - CluhKO mice probed with indicated antibodies . GAPDH was used as loading control . E , F Quantification of western blots as shown in ( D ) . Antibody signal was normalized to GAPDH signal and signal of phospho - protein was normalized to signal of the total protein ( n = 8 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ( Student ' s t - test ) . ( H , J ) * , P ≤ 0 . 05 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . G Confocal images of primary hepatocytes grown in HBSS for 16 h and stained with anti - cleaved caspase 3 antibody . Scale bar , 25 µm . H Quantification of cleaved caspase 3 positive hepatocytes treated with or without rapamycin . More than 100 cells per replicate were analyzed in a blind fashion ( n = 3 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . Error bars show minimum and maximum values . I IHC staining of cleaved caspase 3 in liver sections of starved Li - CluhWT and Li - CluhKO mice . Arrows point to positive apoptotic foci . Scale bar , 200 µm . J Quantification of positive apoptotic foci The number of positive spots was relativized to the area analyzed ( n = 5 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM . K . Confocal images of primary hepatocytes grown in indicated media and stained with anti - G3BP1 and anti - mTOR . Scale bar , 10 µm . L Manders ' colocalization coefficient between G3BP1 and mTOR staining ( n > 50 cells isolated from 3 mice ) . Data information : In ( L ) , data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * * * , P ≤ 0 . 001 ( Student ' s t - test ) . ( H , J ) * , P ≤ 0 . 05 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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

Figure 6A Representative western blot of primary hepatocytes starved in HBSS for the indicated time points and probed with indicated antibodies. Pan-actin was used as loading control. B, C Quantification of western blots as shown in (A). Antibody signal was normalized to pan-actin signal and signal of phospho-protein was normalized to signal of total protein (n=4 independent experiments). Data information: In (B, C), data shows the mean ± SEM. **, P≤0.01 for genotype and ***, P≤0.001 for time (Two-way ANOVA). Error bars show minimum and maximum values.D Representative western blot of total protein extracts from livers of starved Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. GAPDH was used as loading control. E, F Quantification of western blots as shown in (D). Antibody signal was normalized to GAPDH signal and signal of phospho-protein was normalized to signal of the total protein (n=8 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM. Error bars show minimum and maximum values. ***, P≤0.001 (Student's t-test). (H, J) *, P≤0.05; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).G Confocal images of primary hepatocytes grown in HBSS for 16 h and stained with anti-cleaved caspase 3 antibody. Scale bar, 25 µm.H Quantification of cleaved caspase 3 positive hepatocytes treated with or without rapamycin. More than 100 cells per replicate were analyzed in a blind fashion (n=3 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. Error bars show minimum and maximum values.I IHC staining of cleaved caspase 3 in liver sections of starved Li-CluhWT and Li-CluhKO mice. Arrows point to positive apoptotic foci. Scale bar, 200 µm.J Quantification of positive apoptotic foci The number of positive spots was relativized to the area analyzed (n=5 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM.K. Confocal images of primary hepatocytes grown in indicated media and stained with anti-G3BP1 and anti-mTOR. Scale bar, 10 µm. L Manders' colocalization coefficient between G3BP1 and mTOR staining (n >50 cells isolated from 3 mice). Data information: In (L), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. ***, P≤0.001 (Student's t-test). (H, J) *, P≤0.05; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


Figure 7A Confocal images of liver cryosections from Li - CluhWT and Li - CluhKO - LC3 - GFP mice . Scale bar , 25 µm . B Quantification of LC3 signal morphology of images shown in ( A ) . Graphs show relative number of LC3 autophagosomes and the relative area occupied per nuclei . 4 images were quantified per animal and averaged . Nuclear staining was excluded for the analysis ( n = 4 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t - test ) . C Confocal images of primary hepatocytes derived from Li - CluhWT and Li - CluhKO - LC3 - GFP mice . Hepatocytes were starved for 2 h in HBSS . Scale bar , 10μm . D - E Quantification of LC3 puncta number ( D ) and area ( E ) per cell ( n ≥ 100 cells isolated from 3 mice ) . Data information : In ( D , E ) , data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . F Western blot of total protein extracts from livers of Li - CluhWT and Li - CluhKO mice probed with LAMP1 . GAPDH was used as loading control . G Quantification of western blot shown in ( F ) . WT : n = 4 ; KO : n = 3 . Data information : In data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t - test ) . H Representative western blot of primary hepatocytes treated for 2 h with bafilomycin A in the indicated media and probed with indicated antibodies . Two different exposure times are shown for LC3 . I refers to total LC3 and II indicates lipidated form of LC3 . Asterisk indicates signal of previous incubation . I Quantification of LC3 levels in western blots as shown in ( H ) ( n = 4 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . J Quantification of autophagic flux of western blot as shown in ( H ) . The flux was calculated as the increase in LC3II upon bafilomycin A treatment in each condition . The flux in KO cells was normalized to flux in WT cells . ( n = 4 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( Student ' s t - test ) . Quantification of p62 levels in western blots ( n = 4 independent experiments ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . Quantification of CLUH levels in western blots ( n = 4 independent experiments ) . Data information data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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

Figure 7A Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO-LC3-GFP mice. Scale bar, 25 µm. B Quantification of LC3 signal morphology of images shown in (A). Graphs show relative number of LC3 autophagosomes and the relative area occupied per nuclei. 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=4 mice per genotype). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test).C Confocal images of primary hepatocytes derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice. Hepatocytes were starved for 2 h in HBSS. Scale bar, 10μm. D-E Quantification of LC3 puncta number (D) and area (E) per cell (n ≥100 cells isolated from 3 mice). Data information: In (D, E), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).F Western blot of total protein extracts from livers of Li-CluhWT and Li-CluhKO mice probed with LAMP1. GAPDH was used as loading control. G Quantification of western blot shown in (F). WT: n=4; KO: n=3. Data information: In data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test).H Representative western blot of primary hepatocytes treated for 2 h with bafilomycin A in the indicated media and probed with indicated antibodies. Two different exposure times are shown for LC3. I refers to total LC3 and II indicates lipidated form of LC3. Asterisk indicates signal of previous incubation. I Quantification of LC3 levels in western blots as shown in (H) (n=4 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).J Quantification of autophagic flux of western blot as shown in (H). The flux was calculated as the increase in LC3II upon bafilomycin A treatment in each condition. The flux in KO cells was normalized to flux in WT cells. (n=4 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (Student's t-test).Quantification of p62 levels in western blots (n=4 independent experiments). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).Quantification of CLUH levels in western blots (n=4 independent experiments). Data information data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


Figure 8Western blot analysis of total protein extracts from livers of 8 weeks old Li - CluhWT and Li - CluhKO mice probed with indicated antibodies . GAPDH was used as loading control . B Quantification of western blot shown in ( A ) . WT : n = 4 ; KO : n = 3 . Data information data are presented as histograms showing the mean ± SEM . * * * , P ≤ 0 . 001 ; * , P ≤ 0 . 05 ( Student ' s t - test ) . C Western blot analysis of isolated mitochondria from Li - CluhWT and Li - CluhKO mice probed with indicated antibodies . PonceauS staining was used as a loading control . D Quantification of western blot ( n = 3 mice per genotype ) . Data information : , data are presented as histograms showing the mean ± SEM . * * * , P ≤ 0 . 001 ; * , P ≤ 0 . 05 ( Student ' s t - test ) . E Confocal images of liver cryosections derived from Li - CluhWT and Li - CluhKO - LC3 - GFP mice and stained with anti - TOM20 antibody . On the right side , indicated boxes were 3 . 5x magnified . Arrows point to colocalizing spots . Scale bar , 25 μm . F Manders ' colocalization coefficient between TOM20 and LC3 from experiments shown in ( E ) . 5 images were analyzed per animal to get an averaged value ( n = 5 mice per genotype ) . Data information , data are presented as histograms showing the mean ± SEM . * * * , P ≤ 0 . 001 ; * , P ≤ 0 . 05 ( Student ' s t - test ) . G Confocal images of primary hepatocytes derived from Li - CluhWT and Li - CluhKO - LC3 - GFP mice and stained with anti - TOM20 antibody . When indicated cells were treated with rapamycin ( 200 nM for 4h ) . Bottom panels show 2 . 3x magnified areas for each channel of indicated boxes . Arrows point to colocalizing spots . Scale bar , 10 μm . H Manders ´ colocalization coefficient from experiment shown in ( G ) ( n ≥ 50 cells isolated from 3 mice per genotype ) . Data information : In ( H ) , data are presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . I Confocal images of WT and Cluh KO MEFs transfected with mCherry - GFP - FIS101 - 152 and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A / oligomycin . Arrows indicate mitophagosomes . Scale bar , 10μm . J Quantification of percentage of cells with mitophagosomes . Cells were considered positive when red signal was clearly recognizable ( n = 3 - 5 independent experiments , > 100 cells per experiment ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) . K Confocal images of WT and Cluh KO MEFs transfected with mCherry - GFP - FIS101 - 152 , incubated with 200 nM rapamycin and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A / oligomycin . Arrows indicate mitophagosomes . Scale bar , 10 μm . L Quantification of percentage of cells with mitophagosomes . Cells were considered positive when red signal was clearly recognizable ( n = 3 - 5 independent experiments , > 100 cells per experiment ) . Data information : data are presented as histograms showing the mean ± SEM . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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

Figure 8Western blot analysis of total protein extracts from livers of 8 weeks old Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. GAPDH was used as loading control. B Quantification of western blot shown in (A). WT: n=4; KO: n=3. Data information data are presented as histograms showing the mean ± SEM. ***, P≤0.001; *, P≤0.05 (Student's t-test).C Western blot analysis of isolated mitochondria from Li-CluhWT and Li-CluhKO mice probed with indicated antibodies. PonceauS staining was used as a loading control.D Quantification of western blot (n=3 mice per genotype). Data information: , data are presented as histograms showing the mean ± SEM. ***, P≤0.001; *, P≤0.05 (Student's t-test).E Confocal images of liver cryosections derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice and stained with anti-TOM20 antibody. On the right side, indicated boxes were 3.5x magnified. Arrows point to colocalizing spots. Scale bar, 25 μm. F Manders' colocalization coefficient between TOM20 and LC3 from experiments shown in (E). 5 images were analyzed per animal to get an averaged value (n=5 mice per genotype). Data information , data are presented as histograms showing the mean ± SEM. ***, P≤0.001; *, P≤0.05 (Student's t-test).G Confocal images of primary hepatocytes derived from Li-CluhWT and Li-CluhKO-LC3-GFP mice and stained with anti-TOM20 antibody. When indicated cells were treated with rapamycin (200 nM for 4h). Bottom panels show 2.3x magnified areas for each channel of indicated boxes. Arrows point to colocalizing spots. Scale bar, 10 μm. H Manders´ colocalization coefficient from experiment shown in (G) (n ≥ 50 cells isolated from 3 mice per genotype). Data information: In (H), data are presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).I Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101-152 and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A/ oligomycin. Arrows indicate mitophagosomes. Scale bar, 10μm. J Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n= 3-5 independent experiments, >100 cells per experiment). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).K Confocal images of WT and Cluh KO MEFs transfected with mCherry-GFP-FIS101-152, incubated with 200 nM rapamycin and grown for 16 h in galactose medium or in medium containing 10 μM antimycin A/ oligomycin. Arrows indicate mitophagosomes. Scale bar, 10 μm. L Quantification of percentage of cells with mitophagosomes. Cells were considered positive when red signal was clearly recognizable (n= 3-5 independent experiments, >100 cells per experiment). Data information: data are presented as histograms showing the mean ± SEM. *, P≤0.05; **, P≤0.01; ***, P≤0.001 (One-way ANOVA, Tukey's multiple comparisons test).


Figure 9A Confocal images of liver cryosections from Li - CluhWT and Li - CluhKO mice injected with rapamycin or mock solution and stained with anti - TOM20 antibody . Scale bar , 25 µm . B Quantification of morphological parameters of experiments shown in ( A ) . 4 images were quantified per animal and averaged . Nuclear staining was excluded for the analysis ( n = 3 mice per genotype ) . Data information : data are presented as histograms showing the mean ± SEM . * * , P ≤ 0 . 01 ; * , P ≤ 0 . 05 ( Student ' s t - test ) . C Confocal images of liver cryosections from Li - CluhWT and Li - CluhKO mice injected with rapamycin or mock solution and stained with anti - LAMP1 antibody . Scale bar , 25 µm . D Quantification of morphological parameters of experiments shown in ( C ) . 4 images were quantified per animal and averaged . Nuclear staining was excluded for the analysis ( n = 3 mice per genotype ) . Data information : , data are presented as histograms showing the mean ± SEM . * * , P ≤ 0 . 01 ; * , P ≤ 0 . 05 ( Student ' s t - test ) . E Confocal images of COS - 7 cells downregulated for the indicated genes and stained with anti - TOM20 antibody . Scale bar , 10 µm . F Representative western blot of COS - 7 cells downregulated for the indicated genes of experiments and probed with indicated antibodies . Asterisks indicate unspecific signal or signal of previous incubation . G Quantification of morphological parameters of experiments ( n > 100 cells from 3 independent experiments ) . Data information : In ( G ) , data presented as boxplots showing the median , the first and the third quartile . Error bars show minimum and maximum values . ( G ) * * * , P ≤ 0 . 001 ; * * , P ≤ 0 . 01 * , P ≤ 0 . 05 ( One - way ANOVA , Tukey ' s multiple comparisons test ) .

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

Figure 9A Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-TOM20 antibody. Scale bar, 25 µm. B Quantification of morphological parameters of experiments shown in (A). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype).Data information: data are presented as histograms showing the mean ± SEM. **, P≤0.01; *, P≤0.05 (Student's t-test).C Confocal images of liver cryosections from Li-CluhWT and Li-CluhKO mice injected with rapamycin or mock solution and stained with anti-LAMP1 antibody. Scale bar, 25 µm. D Quantification of morphological parameters of experiments shown in (C). 4 images were quantified per animal and averaged. Nuclear staining was excluded for the analysis (n=3 mice per genotype). Data information: , data are presented as histograms showing the mean ± SEM. **, P≤0.01; *, P≤0.05 (Student's t-test).E Confocal images of COS-7 cells downregulated for the indicated genes and stained with anti-TOM20 antibody. Scale bar, 10 µm.F Representative western blot of COS-7 cells downregulated for the indicated genes of experiments and probed with indicated antibodies. Asterisks indicate unspecific signal or signal of previous incubation.G Quantification of morphological parameters of experiments (n>100 cells from 3 independent experiments). Data information: In (G), data presented as boxplots showing the median, the first and the third quartile. Error bars show minimum and maximum values. (G) ***, P≤0.001; **, P≤0.01 *, P≤0.05 (One-way ANOVA, Tukey's multiple comparisons test).


Figure 1 ( A , B ) Expression of UVRAG under cell stress . HL60 and HCT116 cells were treated with doxorubicin ( 1 μg / ml ) , cisplatin ( 10 μm ) and UV irradiation ( 5 min after 50 mJ / cm2 ) for the indicated time , and then apoptosis , autophagy and fluorescence intensity of UVRAG was assayed . Scale bar , 10 μm . ( C ) In parallel , the total protein extracts were used for western blot analysis . ( D , E ) Pharmacological inhibition ( for example , 3 ‐ methyladenine ( 3 ‐ MA ) , 10 mM ) or genetic deletion ( for example , ATG5 − / − ) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin ( Doxo ) and UV irradiation ( n = 3 , * P0 . 05 ) . AU , arbitrary units ; C ‐ PARP , cleaved poly ‐ ADP ribose polymerase ; LC3 , light chain 3 ; mRNA , messenger RNA ; PI , propidium iodide ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance ‐ associated gene .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,B) Expression of UVRAG under cell stress. HL60 and HCT116 cells were treated with doxorubicin (1 μg/ml), cisplatin (10 μm) and UV irradiation (5 min after 50 mJ/cm2) for the indicated time, and then apoptosis, autophagy and fluorescence intensity of UVRAG was assayed. Scale bar, 10 μm. (C) In parallel, the total protein extracts were used for western blot analysis.(D,E) Pharmacological inhibition (for example, 3‐methyladenine (3‐MA), 10 mM) or genetic deletion (for example, ATG5−/−) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin (Doxo) and UV irradiation (n=3, *P0.05). AU, arbitrary units; C‐PARP, cleaved poly‐ADP ribose polymerase; LC3, light chain 3; mRNA, messenger RNA; PI, propidium iodide; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance‐associated gene.


Figure 2 ( A - C ) After transfection with UVRAG shRNA or control shRNA for 48 h , HL60 ( A ) , HCT116 ( B ) and HeLa ( C ) cells were treated with doxorubicin ( Doxo , 1 μg / ml ) , cisplatin ( Cis , 10 μm ) and UV irradiation ( 5 min after 50 mJ / cm2 ) for 12 h or starvation ( HBSS ) for 3 h , and then apoptosis and autophagy were assayed ( n = 3 , * P0 . 05 ) . ( D ) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A ( 10 μg / ml ) and E64D ( 10 μg / ml ) after doxorubicin ( 1 μg / ml ) treatment for 12 h . * P0 . 05 compared with control shRNA group . ( E ) After transfection with UVRAG shRNA or control shRNA for 48 h , ATG5 + / + and ATG5 − / − murine embryonic fibroblasts ( MEFs ) were treated with doxorubicin ( 1 μg / ml ) , with or without Z ‐ VAD ‐ FMK ( 20 μM ) for 12 h , and then apoptosis was assayed ( n = 3 , * P0 . 05 ) . ( F ) Nude mice were inoculated with 4 × 106 HL60 tumour cells following transfection of control ( shControl ) or UVRAG ( shUVRAG ) ‐ specific shRNA and treated with doxorubicin ( 10 mg / kg ) beginning at day 7 . Tumours were measured twice weekly , and volumes were calculated for 21 days ( n = 10 , * P0 . 05 ; shControl + Doxo compared with shUVRAG + Doxo ) . ( G ) On day 21 in the experiments described in ( F ) , apoptosis and UVRAG expression in tumour samples were assayed by TUNEL or western blotting , respectively ( n = 10 , * P0 . 05 ) . BAX , BCL2 ‐ associated X protein ; HBSS , Hank ' s Buffered Salt Solution ; LC3 , light chain 3 ; shRNA , short ‐ hairpin RNA ; TUNEL , terminal deoxytransferase uridine triphosphate nick end labelling ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) After transfection with UVRAG shRNA or control shRNA for 48 h, HL60 (A), HCT116 (B) and HeLa (C) cells were treated with doxorubicin (Doxo, 1 μg/ml), cisplatin (Cis, 10 μm) and UV irradiation (5 min after 50 mJ/cm2) for 12 h or starvation (HBSS) for 3 h, and then apoptosis and autophagy were assayed (n=3, *P0.05).(D) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (10 μg/ml) and E64D (10 μg/ml) after doxorubicin (1 μg/ml) treatment for 12 h. *P0.05 compared with control shRNA group.(E) After transfection with UVRAG shRNA or control shRNA for 48 h, ATG5+/+ and ATG5−/− murine embryonic fibroblasts (MEFs) were treated with doxorubicin (1 μg/ml), with or without Z‐VAD‐FMK (20 μM) for 12 h, and then apoptosis was assayed (n=3, *P0.05).(F) Nude mice were inoculated with 4 × 106 HL60 tumour cells following transfection of control (shControl) or UVRAG (shUVRAG)‐specific shRNA and treated with doxorubicin (10 mg/kg) beginning at day 7. Tumours were measured twice weekly, and volumes were calculated for 21 days (n=10, *P0.05; shControl+Doxo compared with shUVRAG+Doxo).(G) On day 21 in the experiments described in (F), apoptosis and UVRAG expression in tumour samples were assayed by TUNEL or western blotting, respectively (n=10, *P0.05). BAX, BCL2‐associated X protein; HBSS, Hank's Buffered Salt Solution; LC3, light chain 3; shRNA, short‐hairpin RNA; TUNEL, terminal deoxytransferase uridine triphosphate nick end labelling; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


Figure 3 ( A ) Control shRNA or UVRAG shRNA cells were transfected with BAX plasmid , and apoptosis was assayed ( n = 3 , * P0 . 05 ) . ( B ) In parallel , the mitochondrial membrane potential , cytochrome c release from mitochondria , and caspase ‐ 3 and ‐ 9 activation in HL60 cells were analysed ( n = 3 , * P0 . 05 ) . ( C ) After transfection with UVRAG vector or control vector for 48 h , cells were transected or treated with BAX ( 1 μg ) , BAD ( 1 μg ) , BID ( 1 μg ) , FAS antibody ( 10 μg / ml ) or TRAIL ( 100 ng / ml ) for 24 h , then cell apoptosis was analysed ( n = 3 , * P0 . 05 ) . ( D ) Knockdown of UVRAG in BAX − / − HCT116 cells restored the sensitivity to UV treatment ‐ induced apoptosis ( n = 3 , * P0 . 05 ) . AU , arbitrary units ; BAX , BCL2 ‐ associated X protein ; shRNA , short ‐ hairpin RNA ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3(A) Control shRNA or UVRAG shRNA cells were transfected with BAX plasmid, and apoptosis was assayed (n=3, *P0.05).(B) In parallel, the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase‐3 and ‐9 activation in HL60 cells were analysed (n=3, *P0.05).(C) After transfection with UVRAG vector or control vector for 48 h, cells were transected or treated with BAX (1 μg), BAD (1 μg), BID (1 μg), FAS antibody (10 μg/ml) or TRAIL (100 ng/ml) for 24 h, then cell apoptosis was analysed (n=3, *P0.05).(D) Knockdown of UVRAG in BAX−/− HCT116 cells restored the sensitivity to UV treatment‐induced apoptosis (n=3, *P0.05). AU, arbitrary units; BAX, BCL2‐associated X protein; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


Figure 4 ( A , B ) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co ‐ immunoprecipitation ( RIPA or CHAPS buffer ) or western blotting . ( C ) Proteins in the pellet after GST pull ‐ down assays in HCT116 cell lysate were analysed by SDS - PAGE and immunodetected with UVRAG or GST antibodies . ( D ) UVRAG C2 domain deletion terminates BAX binding . HCT116 cells were transfected with Flag ‐ UVRAG or its mutants ( ΔC2 , ΔCCD ) , and then assayed for protein expression levels as indicated by IP or western blot . ( E ) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin ( Doxo , 1 μg / ml ) , UV irradiation ( 5 min after 50 mJ / cm2 ) or transfected with BAX ( 1 μg ) for 24 h . BAX , BCL2 ‐ associated X protein ; CCD , coiled ‐ coil domain ; CHAPS , 3 ‐ [ ( 3 ‐ cholamidopropyl ) ‐ dimethylammonio ] ‐ 1 ‐ propane sulphonate ; GST , glutathione ‐ S ‐ transferase ; IB , immunoblotting ; IgG , immunoglobulin G ; IP , immunoprecipitation ; RIPA , radioimmunoprecipitation assay ; SDS - PAGE , SDS - polyacrylamide gel electrophoresis ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

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

Figure 4(A,B) HL60 and HCT116 cells were treated with UV irradiation for 24 h and then assayed for protein expression levels as indicated by co‐immunoprecipitation (RIPA or CHAPS buffer) or western blotting.(C) Proteins in the pellet after GST pull‐down assays in HCT116 cell lysate were analysed by SDS-PAGE and immunodetected with UVRAG or GST antibodies.(D) UVRAG C2 domain deletion terminates BAX binding. HCT116 cells were transfected with Flag‐UVRAG or its mutants (ΔC2, ΔCCD), and then assayed for protein expression levels as indicated by IP or western blot.(E) Apoptosis was assayed in indicated HCT116 cells after treatment with doxorubicin (Doxo, 1 μg/ml), UV irradiation (5 min after 50 mJ/cm2) or transfected with BAX (1 μg) for 24 h. BAX, BCL2‐associated X protein; CCD, coiled‐coil domain; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate; GST, glutathione‐S‐transferase; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation; RIPA, radioimmunoprecipitation assay; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


Figure 5 UVRAG inhibits mitochondrial translocation of BAX . After transfection with indicated shRNA or cDNA for 48 h , HL60 cells were treated with doxorubicin ( Doxo ) and UV irradiation for 12 h , and then BAX in cytosol ( Cyt ) and mitochondria ( Mit ) was assayed by western blotting ( A - C ) and activation of BAX ( D - F ) was assayed by western blotting after IP using BAX monoclonal antibody ( clone 6A7 ) * P0 . 05 . AU , arbitrary units ; BAX , BCL2 ‐ associated X protein ; CHAPS , 3 ‐ [ ( 3 ‐ cholamidopropyl ) ‐ dimethylammonio ] ‐ 1 ‐ propane sulphonate buffer ; Cyt c , cytochrome c ; IP , immunoprecipitation ; shRNA , short ‐ hairpin RNA ; UV , ultraviolet ; UVRAG , ultraviolet irradiation resistance associated gene .

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

Figure 5 UVRAG inhibits mitochondrial translocation of BAX. After transfection with indicated shRNA or cDNA for 48 h, HL60 cells were treated with doxorubicin (Doxo) and UV irradiation for 12 h, and then BAX in cytosol (Cyt) and mitochondria (Mit) was assayed by western blotting (A-C) and activation of BAX (D-F) was assayed by western blotting after IP using BAX monoclonal antibody (clone 6A7) *P0.05. AU, arbitrary units; BAX, BCL2‐associated X protein; CHAPS, 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulphonate buffer; Cyt c, cytochrome c; IP, immunoprecipitation; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.


Figure 2Effects of ubiquitin surface mutants based on the UbcH7 - Ub : R0RBR : pUb model for the ubiquitin loading and off - loading potential of pParkinCH ( pParkinC431S , H433A ) . Coomassie stained gels ( top ) depict the formation of a pParkinCH ~ Ub oxyester intermediate after 60 minutes in the absence and presence of pUb using the indicated ubiquitin species . Anti - HA blots ( bottom ) depict the ubiquitination of HA - tagged Miro1181 - 579 by pParkin after 10 minutes in the absence and presence of pUb using the indicated ubiquitin species . The E24A substitution in Ub is a negative control as this residue faces away from the interface with parkin . Auto - ubiquitination assay for parkin using ARJP and non - ARJP substitutions in parkin observed near the interface with UbcH7 - Ub . Assays were done in the absence and presence of pUb . Experiments with PINK1 were done by treating parkin : pUb with PINK1 for 30 minutes prior to adding other reagents needed for ubiquitination . Assays were monitored using a DyLight - labelled Ub protein and measuring fluorescence at 800 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Effects of ubiquitin surface mutants based on the UbcH7-Ub:R0RBR:pUb model for the ubiquitin loading and off-loading potential of pParkinCH (pParkinC431S,H433A). Coomassie stained gels (top) depict the formation of a pParkinCH ~ Ub oxyester intermediate after 60 minutes in the absence and presence of pUb using the indicated ubiquitin species. Anti-HA blots (bottom) depict the ubiquitination of HA-tagged Miro1181-579 by pParkin after 10 minutes in the absence and presence of pUb using the indicated ubiquitin species. The E24A substitution in Ub is a negative control as this residue faces away from the interface with parkin.Auto-ubiquitination assay for parkin using ARJP and non-ARJP substitutions in parkin observed near the interface with UbcH7-Ub. Assays were done in the absence and presence of pUb. Experiments with PINK1 were done by treating parkin:pUb with PINK1 for 30 minutes prior to adding other reagents needed for ubiquitination. Assays were monitored using a DyLight-labelled Ub protein and measuring fluorescence at 800 nm.


Figure 5Exposure of the catalytic Cys431 is parkin as assessed by reaction with a UbVS probe . The different stages of activation using parkin , parkin : pUb , pParkin : pUb and pParkin : pUb in combination with an isopeptide linked UbcH7 - Ub conjugate are indicated above each gel . Following addition of UbVS , samples were taken at the times indicated ( 0 - 10 minutes ) and visualized by SDS - PAGE . Relative percentages of pParkin and the pParkin - Ub adduct as a function of time . Intensity percentages were calculated as a function of total intensity of pParkin - Ub , parkin and UbVS / pUb bands . Error bars represent standard deviation from the average for duplicate measurements . Exposure of the catalytic Cys431 is R0RBR parkin as assessed by reaction with a UbVS probe . The different stages of activation using R0RBR , R0RBR : pUb , R0RBR : pUb : pUbl and R0RBR : pUb : pUbl in combination with an isopeptide linked UbcH7 - Ub conjugate are indicated above each gel . Following addition of UbVS , samples were taken at the times indicated ( 0 - 60 minutes ) and visualized by SDS - PAGE Relative percentages of R0RBR and the R0RBR - Ub adduct as a function of time . Intensity percentages were calculated as a function of total intensity of the R0RBR - Ub , R0RBR and UbVS / pUb / pUbl bands .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5Exposure of the catalytic Cys431 is parkin as assessed by reaction with a UbVS probe. The different stages of activation using parkin, parkin:pUb, pParkin:pUb and pParkin:pUb in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-10 minutes) and visualized by SDS-PAGE.   Relative percentages of pParkin and the pParkin-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of pParkin -Ub, parkin and UbVS/pUb bands. Error bars represent standard deviation from the average for duplicate measurements.  Exposure of the catalytic Cys431 is R0RBR parkin as assessed by reaction with a UbVS probe. The different stages of activation using R0RBR, R0RBR:pUb, R0RBR:pUb:pUbl and R0RBR:pUb:pUbl in combination with an isopeptide linked UbcH7-Ub conjugate are indicated above each gel. Following addition of UbVS, samples were taken at the times indicated (0-60 minutes) and visualized by SDS-PAGE   Relative percentages of R0RBR and the R0RBR-Ub adduct as a function of time. Intensity percentages were calculated as a function of total intensity of the R0RBR-Ub, R0RBR and UbVS/pUb/pUbl bands.


Figure 3C - D . ID2 ubiquitination on FANCI results in increased rate of FANCD2 ubiquitination ( B ) , whereas ID2 ubiquitination on FANCD2 results in increased rate of FANCI ubiquitination ( C ) . Protein complexes were assembled in vitro on ice in the presence of dsDNA ( 32 bp ) and their in vitro ubiquitination at 30o C was subsequently monitored in a time - course : at indicative time - points , aliquots of the reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies ( Top ) . For each protein complex , data - points from three replicate experiments ( three technical replicates ) were used in fitting to a one - phase association model ( Bottom ) .

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Figure 3C-D. ID2 ubiquitination on FANCI results in increased rate of FANCD2 ubiquitination (B), whereas ID2 ubiquitination on FANCD2 results in increased rate of FANCI ubiquitination (C). Protein complexes were assembled in vitro on ice in the presence of dsDNA (32 bp) and their in vitro ubiquitination at 30o C was subsequently monitored in a time-course: at indicative time-points, aliquots of the reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies (Top). For each protein complex, data-points from three replicate experiments (three technical replicates) were used in fitting to a one-phase association model (Bottom).


Figure 4A . USP1 - UAF1 - mediated deubiquitination of V5 - FANCI and FANCD2 was assessed in the absence or presence of DNA ( 51 bp ) , when ubiquitinated versions of these proteins were in isolation ( IUb and D2Ub ) or within singly / doubly ubiquitinated ID2 complexes ( IUbD2 , IUbD2Ub and ID2Ub ) . At indicated time - points , aliquots of each reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies . C . Deletion of N - terminus ( ∆ N ) of USP1 ( residues 1 - 54 ) results in greatly reduced FANCD2 deubiquitination . Assays were performed in triplicate ( technical replicates ) as in A , but all reactions contained DNA . Left : Western blotting of reaction products at zero and 30 minutes using FANCD2 and V5 antibodies . Right : Residual FANCI and FANCD2 ubiquitination following USP1 - UAF1 treatment for 30 min . Replicate residual ubiquitination values and statistically significant changes ( one - way ANOVA test with Bonferroni correction ) are shown . * * * p < 0 . 001 .

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

Figure 4A. USP1-UAF1-mediated deubiquitination of V5-FANCI and FANCD2 was assessed in the absence or presence of DNA (51 bp), when ubiquitinated versions of these proteins were in isolation (IUb and D2Ub) or within singly/doubly ubiquitinated ID2 complexes (IUbD2, IUbD2Ub and ID2Ub). At indicated time-points, aliquots of each reaction were removed and analysed by western blotting using FANCD2 and V5 antibodies.C. Deletion of N-terminus (∆N) of USP1 (residues 1-54) results in greatly reduced FANCD2 deubiquitination. Assays were performed in triplicate (technical replicates) as in A, but all reactions contained DNA. Left: Western blotting of reaction products at zero and 30 minutes using FANCD2 and V5 antibodies. Right: Residual FANCI and FANCD2 ubiquitination following USP1-UAF1 treatment for 30 min. Replicate residual ubiquitination values and statistically significant changes (one-way ANOVA test with Bonferroni correction) are shown. ***p<0.001.


Figure 5Alanine mutagenesis of key FANCD2 ' s residues ( H209 , V243 and P244 ) participating in the extended FANCD2 - ubiquitin interface ( IUbD2 - DNA and IUbD2Ub - DNA structures ) , results in enhanced FANCI deubiquitination by USP1 - UAF1 . C . FANCI deubiquitination by USP1 - UAF1 ( incubation with 50 nM USP1 - UAF1 for 30 minutes at room temperature ) in the presence of DNA and , in the absence or presence of wild - type ( D2WT ) or mutant ( D2H209A , VP243AA ) FANCD2 . Replicate residual ubiquitination values ( three technical replicates ) and statistically significant changes ( t - test and one - way ANOVA test with Bonferroni correction ) are shown . D . FANCI was ubiquitinated with DyLight - 680 labelled ubiquitin ( IUb - DL680 ) and its deubiquitination by USP1 - UAF1 ( incubation with 100 nM USP1 - UAF1 for 20 minutes at room temperature ) , was assessed at increasing concentrations ( 20 , 200 or 200 nM ) of , either wild - type ( D2WT ) , or mutant ( D2H209A , VP243AA ) FANCD2 . Experiment was repeated three times ( technical replicates ) , and the fold change in residual FANCI ubiquitination was determined by normalisation to the mean intensity of IUb - DL680 at 20 nM FANCD2 ( average from six values , for both D2WT and D2H209A , VP243AA ) . Mean ( with range ) fold - increase values and statistically significant changes upon FANCD2 mutation for each FANCD2 concentration ( t - test and two - way ANOVA test with Bonferroni correction ) are shown .

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Figure 5Alanine mutagenesis of key FANCD2's residues (H209, V243 and P244) participating in the extended FANCD2-ubiquitin interface (IUbD2-DNA and IUbD2Ub-DNA structures), results in enhanced FANCI deubiquitination by USP1-UAF1. C. FANCI deubiquitination by USP1-UAF1 (incubation with 50 nM USP1-UAF1 for 30 minutes at room temperature) in the presence of DNA and, in the absence or presence of wild-type (D2WT) or mutant (D2H209A, VP243AA) FANCD2. Replicate residual ubiquitination values (three technical replicates) and statistically significant changes (t-test and one-way ANOVA test with Bonferroni correction) are shown.D. FANCI was ubiquitinated with DyLight-680 labelled ubiquitin (IUb-DL680) and its deubiquitination by USP1-UAF1 (incubation with 100 nM USP1-UAF1 for 20 minutes at room temperature), was assessed at increasing concentrations (20, 200 or 200 nM) of, either wild-type (D2WT), or mutant (D2H209A, VP243AA) FANCD2. Experiment was repeated three times (technical replicates), and the fold change in residual FANCI ubiquitination was determined by normalisation to the mean intensity of IUb-DL680 at 20 nM FANCD2 (average from six values, for both D2WT and D2H209A, VP243AA). Mean (with range) fold-increase values and statistically significant changes upon FANCD2 mutation for each FANCD2 concentration (t-test and two-way ANOVA test with Bonferroni correction) are shown.


Figure 1A Quantitative RT - PCR ( RT - qPCR ) results showing relative expression levels of mouse Pabpn1l in somatic tissues , oocytes , and preimplantation embryos . n = 3 biological replicates . Error bars indicate S . E . M . B Western blot results of PABPN1L , BTG4 , and phosphorylated ERK1 / 2 ( pERK1 / 2 ) in oocytes and embryos . Total protein from 400 oocytes or embryos is loaded in each lane . DNA damage - binding protein 1 ( DDB1 ) is blotted as a loading control . C Western blot results of PABPN1L in MII oocytes of wild type ( WT ) and Pabpn1l − / − females . DDB1 is blotted as a loading control . D Cumulative pup numbers of WT and Pabpn1l - / - female mice . n = 5 for each genotype . Error bars , S . E . M . * * * P < 0 . 001 by two - tailed student ' s t - test . E Quantification of preimplantation embryos derived from WT and Pabpn1l - / - females when WT embryos reached the corresponding stages . The number of analyzed embryos is indicated ( n ) . Error bars , S . E . M . * * * P < 0 . 001 by two - tailed Student ' s t - test . F Representative images of the embryos collected from the oviducts of mated WT and Pabpn1l - / - females at the indicated time points after hCG injection . Scale bar = 100 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 1A Quantitative RT-PCR (RT-qPCR) results showing relative expression levels of mouse Pabpn1l in somatic tissues, oocytes, and preimplantation embryos. n = 3 biological replicates. Error bars indicate S.E.M.B Western blot results of PABPN1L, BTG4, and phosphorylated ERK1/2 (pERK1/2) in oocytes and embryos. Total protein from 400 oocytes or embryos is loaded in each lane. DNA damage-binding protein 1 (DDB1) is blotted as a loading control.C Western blot results of PABPN1L in MII oocytes of wild type (WT) and Pabpn1l−/− females. DDB1 is blotted as a loading control.D Cumulative pup numbers of WT and Pabpn1l-/- female mice. n = 5 for each genotype. Error bars, S.E.M. ***P < 0.001 by two-tailed student's t-test.E Quantification of preimplantation embryos derived from WT and Pabpn1l-/- females when WT embryos reached the corresponding stages. The number of analyzed embryos is indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.F Representative images of the embryos collected from the oviducts of mated WT and Pabpn1l-/- females at the indicated time points after hCG injection. Scale bar = 100 μm.


Figure 2A Scatter plot comparing the transcripts of GV oocytes and zygotes from WT and Pabpn1l - / - females . Transcripts that increased or decreased by more than 3 - fold in Pabpn1l - deleted GV oocytes or zygotes are highlighted in red or blue , respectively . D RT - qPCR results for relative expression levels of the indicated maternal transcripts in oocytes and zygotes from WT and Pabpn1l − / − females . n = 3 biological replicates . Error bars , SEM . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 by two - tailed Student ' s t - test . E Poly ( A ) tail assay results showing poly ( A ) - tail length of indicated transcripts in WT and Pabpn1l - deleted oocytes and embryos . The poly ( A ) tails of Actin are used as an internal control . Each sample was prepared from 100 oocytes or embryos . Plots show the averaged relative signal intensity ( y - axis ) and length of the PCR products based on mobility ( x - axis ) .

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Figure 2A Scatter plot comparing the transcripts of GV oocytes and zygotes from WT and Pabpn1l-/- females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted GV oocytes or zygotes are highlighted in red or blue, respectively.D RT-qPCR results for relative expression levels of the indicated maternal transcripts in oocytes and zygotes from WT and Pabpn1l −/− females. n = 3 biological replicates. Error bars, SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test.E Poly(A) tail assay results showing poly(A)-tail length of indicated transcripts in WT and Pabpn1l-deleted oocytes and embryos. The poly(A) tails of Actin are used as an internal control. Each sample was prepared from 100 oocytes or embryos. Plots show the averaged relative signal intensity (y-axis) and length of the PCR products based on mobility (x-axis).


Figure 3A Scatter plot comparing the transcripts of 2 - cell embryos from WT and Pabpn1l - / - females . Transcripts that increased or decreased by more than 3 - fold in Pabpn1l - deleted oocytes or embryos are highlighted in red or blue , respectively . C 5 - Ethynyl uridine ( EU ) fluorescence ( green ) showing RNA transcription in WT and Pabpn1l ♀ − / ♂ + 2 - cell embryos . Some WT embryos were treated with α - amanitin as early as the zygote stage and cultured to the 2 - cell stage . The phosphorylated RNA polymerase II CTD repeat YSPTSPS ( pS2 ) ( red ) is co - stained to label the RNA polymerase II activity . Nuclei are labeled by DAPI ( blue ) . Scale bar = 20 μm . D Quantification of EU and pS2 signals in ( A ) . The numbers of analyzed embryos are indicated ( n ) . Error bars , S . E . M . * * * P < 0 . 001 by two - tailed Student ' s t - test . E Relative intensity of tdTomato signals in ( F ) . The numbers of analyzed embryos are indicated ( n ) . Error bars , S . E . M . * * * P < 0 . 001 by two - tailed Student ' s t - test . F Representative images of MuERV - L : : tdTomato relative to GFP signal in the same embryo when WT embryos reached the corresponding stages . Zygotes were injected with the MuERV - L : : tdTomato reporter plasmid and polyadenylated Gfp mRNA ( as a positive control of microinjection ) , then allowed to develop in vitro . Cultured embryos were imaged at 24 h and 40 h after hCG injection . DIC , differential interference contrast . Scale bar = 100 μm .

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Figure 3A Scatter plot comparing the transcripts of 2-cell embryos from WT and Pabpn1l-/- females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted oocytes or embryos are highlighted in red or blue, respectively.C 5-Ethynyl uridine (EU) fluorescence (green) showing RNA transcription in WT and Pabpn1l♀−/♂+ 2-cell embryos. Some WT embryos were treated with α-amanitin as early as the zygote stage and cultured to the 2-cell stage. The phosphorylated RNA polymerase II CTD repeat YSPTSPS (pS2) (red) is co-stained to label the RNA polymerase II activity. Nuclei are labeled by DAPI (blue). Scale bar = 20 μm.D Quantification of EU and pS2 signals in (A). The numbers of analyzed embryos are indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.E Relative intensity of tdTomato signals in (F). The numbers of analyzed embryos are indicated (n). Error bars, S.E.M. ***P < 0.001 by two-tailed Student's t-test.F Representative images of MuERV-L::tdTomato relative to GFP signal in the same embryo when WT embryos reached the corresponding stages. Zygotes were injected with the MuERV-L::tdTomato reporter plasmid and polyadenylated Gfp mRNA (as a positive control of microinjection), then allowed to develop in vitro. Cultured embryos were imaged at 24 h and 40 h after hCG injection. DIC, differential interference contrast. Scale bar = 100 μm.


Figure 4A RNA immunoprecipitation ( RIP ) results showing the interaction of BTG4 with indicated transcripts , in the presence or absence of PABPN1L ( full length , RRM - deleted , or R171A mutant ) . HeLa cells were co - transfected with plasmids expressing FLAG - tagged PABPN1L and HA - tagged BTG4 for 48 h before immunoprecipitating with an anti - HA antibody . RNAs recovered from the immunoprecipitants were subjected to RT - qPCR of the indicated transcripts . Fold change values of both input and IP samples were normalized by RIP results of HA - BTG4 and FLAG - PABPN1L co - expression groups . n = 3 biological replicates . Error bars , S . E . M . The P - value represents the two - tailed Student ' s t - test comparing the RIP results of BTG4 with the indicated transcripts in the presence of PABPN1L , * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 . Co - IP and western blot results showing interactions between PABPN1L and BTG4 . Lysates from HeLa cells expressing HA - BTG4 ( WT or mutants shown in ( B ) ) and FLAG - PABPN1L were immunoprecipitated with an anti - HA antibody . The immunoprecipitated proteins are detected by western blot with the indicated antibodies . D Co - IP and western blot results showing interactions between PABPN1L and BTG4 . Lysates from HeLa cells expressing HA - BTG4 ( WT or mutants shown in ( B ) ) and FLAG - PABPN1L were immunoprecipitated with an anti - HA antibody . The immunoprecipitated proteins are detected by western blot with the indicated antibodies . E Diagrams of mouse BTG2 and BTG4 constructs , and co - IP results showing that PABPN1L binds to BTG4 but not BTG2 . F Diagrams and co - IP results showing BTG4 binding to PABPN1L . Lysates from HeLa cells expressing HA - BTG4 and FLAG - PABPN1L ( full length and C - terminal deleted ) were immunoprecipitated with an anti - FLAG antibody . The immunoprecipitated proteins are detected by western blot with the indicated antibodies . G Diagrams of PABPN1 and PABPN1L constructs , and co - IP results showing that PABPN1L , but not PABPN1 , binds to BTG4 .

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

Figure 4A RNA immunoprecipitation (RIP) results showing the interaction of BTG4 with indicated transcripts, in the presence or absence of PABPN1L (full length, RRM-deleted, or R171A mutant). HeLa cells were co-transfected with plasmids expressing FLAG-tagged PABPN1L and HA-tagged BTG4 for 48 h before immunoprecipitating with an anti-HA antibody. RNAs recovered from the immunoprecipitants were subjected to RT-qPCR of the indicated transcripts. Fold change values of both input and IP samples were normalized by RIP results of HA-BTG4 and FLAG-PABPN1L co-expression groups. n = 3 biological replicates. Error bars, S.E.M. The P-value represents the two-tailed Student's t-test comparing the RIP results of BTG4 with the indicated transcripts in the presence of PABPN1L, *P < 0.05, **P < 0.01, and ***P < 0.001.Co-IP and western blot results showing interactions between PABPN1L and BTG4. Lysates from HeLa cells expressing HA-BTG4 (WT or mutants shown in (B)) and FLAG-PABPN1L were immunoprecipitated with an anti-HA antibody. The immunoprecipitated proteins are detected by western blot with the indicated antibodies.D Co-IP and western blot results showing interactions between PABPN1L and BTG4. Lysates from HeLa cells expressing HA-BTG4 (WT or mutants shown in (B)) and FLAG-PABPN1L were immunoprecipitated with an anti-HA antibody. The immunoprecipitated proteins are detected by western blot with the indicated antibodies.E Diagrams of mouse BTG2 and BTG4 constructs, and co-IP results showing that PABPN1L binds to BTG4 but not BTG2.F Diagrams and co-IP results showing BTG4 binding to PABPN1L. Lysates from HeLa cells expressing HA-BTG4 and FLAG-PABPN1L (full length and C-terminal deleted) were immunoprecipitated with an anti-FLAG antibody. The immunoprecipitated proteins are detected by western blot with the indicated antibodies.G Diagrams of PABPN1 and PABPN1L constructs, and co-IP results showing that PABPN1L, but not PABPN1, binds to BTG4.


Figure 5B RT - qPCR results showing the relative expression level changes ( MII / GV ) of indicated transcripts . Fully grown GV oocytes of Pabpn1l - / - mice were microinjected with mRNAs encoding PABPN1L or BTG4 and were released from meiotic arrest at 12 h after microinjection . Total RNA was extracted from 10 oocytes in each sample . Error bars , S . E . M . * P < 0 . 05 , * * P < 0 . 01 , and * * * P < 0 . 001 by two - tailed Student ' s t - test compared to the first column . ns : non - significant . C Western blot results showing the expression of exogenous PABPN1L ( full length , RRM - deleted , or R171A mutants ) and BTG4 in Pabpn1l - / - oocytes at the MII stage . The total protein from 100 oocytes is loaded in each lane . pERK1 / 2 is blotted to indicate the developmental stages . DDB1 is blotted as a loading control . At least three independent experiments were done with consistent results .

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Figure 5B RT-qPCR results showing the relative expression level changes (MII/GV) of indicated transcripts. Fully grown GV oocytes of Pabpn1l-/- mice were microinjected with mRNAs encoding PABPN1L or BTG4 and were released from meiotic arrest at 12 h after microinjection. Total RNA was extracted from 10 oocytes in each sample. Error bars, S.E.M. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test compared to the first column. ns: non-significant.C Western blot results showing the expression of exogenous PABPN1L (full length, RRM-deleted, or R171A mutants) and BTG4 in Pabpn1l-/- oocytes at the MII stage. The total protein from 100 oocytes is loaded in each lane. pERK1/2 is blotted to indicate the developmental stages. DDB1 is blotted as a loading control. At least three independent experiments were done with consistent results.