Figure 9 A Boxplots of average expression ( log10 ( FPKMs + 1 ) ) for genes sublist 1 - 5 ( lowest to highest expression ) for wild - type ( WT , N = 3 ) mice ( left ) . Pie charts of gene distribution among generated sublists ( 1 - 5 ) for all genes ( middle ) and genes in LADs ( right ) in WT mice . The bottom and top of the boxes are the first and third quartiles , and the line within represents the median . The whiskers denote the interval within 1 . 5 times the interquartile range ( IQR ) from the median . B Average gene expression ( FPKMs ) of genes found in common and wild - type ( WT , N = 3 ) specific LADs for WT ( N = 9 ) and R6 / 1 ( N = 9 ) mice . Each point corresponds to the value from an individual sample . Data is shown as the mean ± S . E . M . * P < 0 . 05 as compared to WT mice ( two - tailed unpaired Student ' s t test ) . Exact P values are reported in Appendix Table S3 . C Average chromatin accessibility ( normalized read counts ) of genes found in common and wild - type ( WT , N = 3 ) specific LADs for WT ( N = 3 ) and R6 / 1 ( N = 3 ) mice . Each point corresponds to the value from an individual sample . Data is shown as the mean ± S . E . M . Wilcoxon Mann Whitney test was used for statistical analysis . Exact P values are reported in Appendix Table S3 . D Venn diagram showing the total number of genes found in common and wild - type ( WT ) specific LADs regions ( left ) . Bar graph showing the number of up - and down - regulated genes found in common and WT specific LADs regions filtering only according to adjusted P - value ( adjusted P - value < 0 . 001 ) or additionally with fold change ( adjusted P - value < 0 . 001 , | FC | > 2 ) obtained from deseq2 differential expression analysis ( see methods ) . E UCSC genome browser capture of wild - type ( WT , N = 3 ) and R6 / 1 ( N = 3 ) mice hippocampus ATAC - seq data , lamin B1 ChIP - seq ( log ( Lb1 ChIP / Input ) ) for WT ( N = 3 ) and R6 / 1 ( N = 3 ) mice hippocampus , hippocampal H3K9ac , CTCF and H3K9me3 for WT mice hippocampus in Fos locus ( left ) . Enhancer regions ( E1 - E5 ) are indicated with arrows . Boxplot of lamin B1 enrichment ( log10 ( Lb1 ChIP / Input ) ) for regions with unchanged , decreased ( closed in R6 / 1 ) and increased ( opened in R6 / 1 ) chromatin accessibility in R6 / 1 ( N = 3 ) mice hippocampus . * P < 0 . 05 as compared to WT ( N = 3 ) mice . ( Wilcoxon Mann Whitney test ) . Exact P values are reported in Appendix Table S3 . The bottom and top of the boxes are the first and third quartiles , and the line within represents the median . The whiskers denote the interval within 1 . 5 times the interquartile range ( IQR ) from the median .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 9 A Boxplots of average expression (log10(FPKMs +1)) for genes sublist 1-5 (lowest to highest expression) for wild-type (WT, N=3) mice (left). Pie charts of gene distribution among generated sublists (1-5) for all genes (middle) and genes in LADs (right) in WT mice. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median. B Average gene expression (FPKMs) of genes found in common and wild-type (WT, N=3) specific LADs for WT (N=9) and R6/1 (N=9) mice. Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. * P < 0.05 as compared to WT mice (two-tailed unpaired Student's t test). Exact P values are reported in Appendix Table S3.  C Average chromatin accessibility (normalized read counts) of genes found in common and wild-type (WT, N=3) specific LADs for WT (N=3) and R6/1 (N=3) mice. Each point corresponds to the value from an individual sample. Data is shown as the mean ± S.E.M. Wilcoxon Mann Whitney test was used for statistical analysis. Exact P values are reported in Appendix Table S3. D Venn diagram showing the total number of genes found in common and wild-type (WT) specific LADs regions (left). Bar graph showing the number of up- and down-regulated genes found in common and WT specific LADs regions filtering only according to adjusted P-value (adjusted P-value <0.001) or additionally with fold change (adjusted P-value <0.001, |FC| > 2) obtained from deseq2 differential expression analysis (see methods). E UCSC genome browser capture of wild-type (WT, N=3) and R6/1 (N=3) mice hippocampus ATAC-seq data, lamin B1 ChIP-seq (log(Lb1 ChIP/Input)) for WT (N=3) and R6/1 (N=3) mice hippocampus, hippocampal H3K9ac, CTCF and H3K9me3 for WT mice hippocampus in Fos locus (left). Enhancer regions (E1-E5) are indicated with arrows. Boxplot of lamin B1 enrichment (log10(Lb1 ChIP/Input)) for regions with unchanged, decreased (closed in R6/1) and increased (opened in R6/1) chromatin accessibility in R6/1 (N=3) mice hippocampus. * P < 0.05 as compared to WT (N=3) mice. (Wilcoxon Mann Whitney test). Exact P values are reported in Appendix Table S3. The bottom and top of the boxes are the first and third quartiles, and the line within represents the median. The whiskers denote the interval within 1.5 times the interquartile range (IQR) from the median.


Figure 10B , C Graphs show the percentage of time exploring each object with respect to the total exploration time in the ( B ) NOLT and ( C ) NORT , 5 weeks after treatment ( Veh , vehicle ; BA , betulinic acid ; WT , wild - type ) . * * * * P < 0 . 0001 compared to the corresponding old location / object . WT veh N = 13 ; R6 / 1 veh N = 10 ; R6 / 1 + BA N = 10 . D Accelerating rotarod was assessed after 7 weeks of treatment . * * P < 0 . 01 and * * * P < 0 . 001 compared to vehicle - treated wild - type ( WT ) mice ; # P < 0 . 05 compared to vehicle - treated R6 / 1 mice . WT veh N = 13 ; R6 / 1 veh N = 9 ; R6 / 1 + BA N = 9 . E Lamin B1 levels were analysed by Western blot in the striatum ( WT veh N = 10 ; R6 / 1 veh N = 6 ; R6 / 1 + BA N = 7 ) , hippocampus ( WT veh N = 12 ; R6 / 1 veh N = 8 ; R6 / 1 + BA N = 7 ) and cortex ( WT veh N = 11 ; R6 / 1 veh N = 6 ; R6 / 1 + BA N = 7 ) after 12 weeks of treatment . * P < 0 . 05 , * * P < 0 . 05 compared to vehicle - treated wild - type ( WT ) mice and # P < 0 . 05 compared to vehicle - treated R6 / 1 mice . Representative immunoblots of lamin B1 and α - tubulin ( as loading control ) for each treatment group are shown . F Lamin B1 intensity and nuclear morphology were analysed by FANSI in hippocampal CA1 neurons nuclei from wild - type ( WT ) and R6 / 1 mice after 12 weeks of treatment . * P < 0 . 05 compared to vehicle - treated WT mice . * P < 0 . 05 as compared to vehicle - treated WT mice . Lamin B1 intensity : WT veh N = 6 ; R6 / 1 veh N = 5 ; R6 / 1 + BA N = 4 ; Lamin B1 circularity : WT veh N = 6 ; R6 / 1 veh N = 5 ; R6 / 1 + BA N = 5 . G Nuclear permeability was analysed by FRAP in hippocampal CA1 neurons nuclei after 12 weeks of treatment . * p < 0 . 05 compared to vehicle - treated wild - type ( WT ) mice . WT veh N = 4 ; R6 / 1 veh N = 5 ; R6 / 1 + BA N = 6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 10B,C Graphs show the percentage of time exploring each object with respect to the total exploration time in the (B) NOLT and (C) NORT, 5 weeks after treatment (Veh, vehicle; BA, betulinic acid; WT, wild-type). ****P < 0.0001 compared to the corresponding old location/object. WT veh N = 13; R6/1 veh N = 10; R6/1+BA N = 10.D Accelerating rotarod was assessed after 7 weeks of treatment. **P < 0.01 and ***P < 0.001 compared to vehicle-treated wild-type (WT) mice; #P < 0.05 compared to vehicle-treated R6/1 mice. WT veh N = 13; R6/1 veh N = 9; R6/1+BA N = 9.E Lamin B1 levels were analysed by Western blot in the striatum (WT veh N = 10; R6/1 veh N = 6; R6/1+BA N = 7), hippocampus (WT veh N = 12; R6/1 veh N = 8; R6/1+BA N = 7) and cortex (WT veh N = 11; R6/1 veh N = 6; R6/1+BA N = 7) after 12 weeks of treatment. *P < 0.05, **P < 0.05 compared to vehicle-treated wild-type (WT) mice and #P < 0.05 compared to vehicle-treated R6/1 mice. Representative immunoblots of lamin B1 and α-tubulin (as loading control) for each treatment group are shown. F Lamin B1 intensity and nuclear morphology were analysed by FANSI in hippocampal CA1 neurons nuclei from wild-type (WT) and R6/1 mice after 12 weeks of treatment. *P < 0.05 compared to vehicle-treated WT mice. *P < 0.05 as compared to vehicle-treated WT mice. Lamin B1 intensity: WT veh N = 6; R6/1 veh N = 5; R6/1+BA N = 4; Lamin B1 circularity: WT veh N = 6; R6/1 veh N = 5; R6/1+BA N = 5.  G Nuclear permeability was analysed by FRAP in hippocampal CA1 neurons nuclei after 12 weeks of treatment. *p < 0.05 compared to vehicle-treated wild-type (WT) mice. WT veh N = 4; R6/1 veh N = 5; R6/1+BA N = 6.


Figure 1 ( B ) Numbers of surface tumors per liver were counted . ( C ) The cumulative diameter of all surface tumors per liver per mouse reflects the overall tumor load . ( D ) Weight of the livers including tumor mass relative to the body weight ( LW / BW ratio ) was recorded . ( E ) Representative pictures of total livers with tumors of the indicated genotypes .

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

Figure 1(B) Numbers of surface tumors per liver were counted. (C) The cumulative diameter of all surface tumors per liver per mouse reflects the overall tumor load. (D) Weight of the livers including tumor mass relative to the body weight (LW/BW ratio) was recorded.(E) Representative pictures of total livers with tumors of the indicated genotypes.


Figure 2 ( A ) Immunoblots of murine wt non - tumorous and tumor tissue lysates were probed for CASP2 , RAIDD , and p53 . HSP90 and PonceauS protein staining served as loading controls . ( B ) Proliferation of tumor tissue was determined by the percentage of Ki67 - positive nuclei analyzed by flow cytometry . T : wt n = 17 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 8 ; NT : wt n = 12 , Casp - 2 - / - n = 11 , Raidd - / - n = 7 , Pidd1 - / - n = 5 . N - numbers refer to biological replicates . ( D ) Immunoblots of the experiment described in ( C ) at t ( 8h ) , t ( 48h ) , and t ( weaning + 5d ) for CASP2 , RAIDD , p53 , γH2AX , and HSP90 as loading control . ( E ) The number of binucleated hepatocytes per field was counted on H & E stained paraffin sections of PBS treated wt and Pidd1 - / - mice at t ( 0h ) and at t ( 48h ) after PBS or DEN treatment . N - numbers refer to biological replicates . ( F ) Immunofluorescence of the same livers was perfomed for the distal centriolar protein CP110 to stain centrioles , β - catenin for cell borders , and DAPI to stain DNA . Representative images of a mononucleated hepatocyte with 2 centrioles and a binucleated hepatocyte with 4 centrioles is shown . ( G ) Centriole number was quantified with respect to the number of nuclei per cell in wt and Pidd1 - / - livers at the time points described in ( E ) ; n = 3 biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O 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) Immunoblots of murine wt non-tumorous and tumor tissue lysates were probed for CASP2, RAIDD, and p53. HSP90 and PonceauS protein staining served as loading controls.(B) Proliferation of tumor tissue was determined by the percentage of Ki67-positive nuclei analyzed by flow cytometry. T: wt n=17, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=8; NT: wt n=12, Casp-2-/- n=11, Raidd-/- n=7, Pidd1-/- n=5. N-numbers refer to biological replicates.(D) Immunoblots of the experiment described in (C) at t(8h), t(48h), and t(weaning+5d) for CASP2, RAIDD, p53, γH2AX, and HSP90 as loading control.(E) The number of binucleated hepatocytes per field was counted on H&E stained paraffin sections of PBS treated wt and Pidd1-/- mice at t(0h) and at t(48h) after PBS or DEN treatment. N-numbers refer to biological replicates.(F) Immunofluorescence of the same livers was perfomed for the distal centriolar protein CP110 to stain centrioles, β-catenin for cell borders, and DAPI to stain DNA. Representative images of a mononucleated hepatocyte with 2 centrioles and a binucleated hepatocyte with 4 centrioles is shown.(G) Centriole number was quantified with respect to the number of nuclei per cell in wt and Pidd1-/- livers at the time points described in (E); n=3 biological replicates.


Figure 3 ( A ) Binucleation was assessed blinded in H & E stained sections of murine non - tumor ( NT ) and tumor tissue ( T ) T : wt n = 14 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 10 ; NT : wt n = 8 , Casp - 2 - / - n = 10 , Raidd - / - n = 8 , Pidd1 - / - n = 9 . N - numbers refer to biological replicates . Binucleation was assessed blinded in H & E stained sections of murine non - tumor ( NT ) and tumor tissue ( T ) also shown in ( B ) . T : wt n = 14 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 10 ; NT : wt n = 8 , Casp - 2 - / - n = 10 , Raidd - / - n = 8 , Pidd1 - / - n = 9 . N - numbers refer to biological replicates . C , D ( C ) Representative histograms of nuclei stained for DNA , which were isolated from non - tumor and tumor tissue of wt and Casp2 - / - mice . These animals are marked as red " x " in the quantification shown in ( D ) were the degree of polyploidy is reflected as percentage of octaploid nuclei ( T : wt n = 17 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 8 ; NT : wt n = 12 , Casp - 2 - / - n = 11 , Raidd - / - n = 7 , Pidd1 - / - n = 5 ) . N - numbers refer to biological replicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Binucleation was assessed blinded in H&E stained sections of murine non-tumor (NT) and tumor tissue (T) T: wt n=14, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=10; NT: wt n=8, Casp-2-/- n=10, Raidd-/- n=8, Pidd1-/- n=9. N-numbers refer to biological replicates.Binucleation was assessed blinded in H&E stained sections of murine non-tumor (NT) and tumor tissue (T) also shown in (B). T: wt n=14, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=10; NT: wt n=8, Casp-2-/- n=10, Raidd-/- n=8, Pidd1-/- n=9. N-numbers refer to biological replicates.C, D (C) Representative histograms of nuclei stained for DNA, which were isolated from non-tumor and tumor tissue of wt and Casp2-/- mice. These animals are marked as red "x" in the quantification shown in (D) were the degree of polyploidy is reflected as percentage of octaploid nuclei (T: wt n=17, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=8; NT: wt n=12, Casp-2-/- n=11, Raidd-/- n=7, Pidd1-/- n=5). N-numbers refer to biological replicates.


Figure 4Copy number variations were determined using whole genome sequencing of 30 nuclei " mini - bulks " isolated from Casp2 - / - or wt tumors ( n = 11 per genotype ) . Diploid and tetraploid pools of tumor cell nuclei were analyzed separately . A , B ( A ) Representative dot plots show the degree of aneuploidy and heterogeneity and are quantified in ( B , empty symbols ) . Data are represented as median , statistical significance determined by one - way ANOVA with multiplicity correction ( Sidak - Holm ) ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 . N - numbers refer to biological replicates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Copy number variations were determined using whole genome sequencing of 30 nuclei "mini-bulks" isolated from Casp2-/- or wt tumors (n=11 per genotype). Diploid and tetraploid pools of tumor cell nuclei were analyzed separately. A, B (A) Representative dot plots show the degree of aneuploidy and heterogeneity and are quantified in (B, empty symbols). Data are represented as median, statistical significance determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01. N-numbers refer to biological replicates.


Figure 5 ( A ) Matched human patient non - tumor ( NT ) and tumor tissue ( T ) samples were analyzed by immunoblotting for CASP2 , PIDD1 and RAIDD on membranes a and b . PonceauS protein staining serves as loading control . The asterisk refers to an unspecific band , " C " and " CC " indicate the functionally active fragments of PIDD1 generated by autoprocessing , PIDD - C and PIDD - CC . Densitometric quantification of these blots is shown in Fig . EV4A . ( B ) mRNA expression analysis of the PIDDosome components in the TCGA Provisional LIHC data set across disease stages . Patients are divided based on the p53 mutation / deletion ( mut / del ) status . Statistical significance was determined using two - way ANOVA with Tukey ' s post - hoc analysis . The central band represents the median , the boxes indicate the 25th and 75th percentile , the whiskers are the 5th and 95th percentiles , statistical significance was defined as * p ≤ 0 . 05 , * * p ≤ 0 . 01 . ( C ) The same data set was analyzed for recurrence - free survival based on high or low expression of CASP2 , RAIDD or PIDD1 divided at the median . Statistical significance was determined using a Log - rank 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Matched human patient non-tumor (NT) and tumor tissue (T) samples were analyzed by immunoblotting for CASP2, PIDD1 and RAIDD on membranes a and b. PonceauS protein staining serves as loading control. The asterisk refers to an unspecific band, "C" and "CC" indicate the functionally active fragments of PIDD1 generated by autoprocessing, PIDD-C and PIDD-CC. Densitometric quantification of these blots is shown in Fig. EV4A.(B) mRNA expression analysis of the PIDDosome components in the TCGA Provisional LIHC data set across disease stages. Patients are divided based on the p53 mutation/deletion (mut/del) status. Statistical significance was determined using two-way ANOVA with Tukey's post-hoc analysis. The central band represents the median, the boxes indicate the 25th and 75th percentile, the whiskers are the 5th and 95th percentiles, statistical significance was defined as * p≤0.05, ** p≤0.01.(C) The same data set was analyzed for recurrence-free survival based on high or low expression of CASP2, RAIDD or PIDD1 divided at the median. Statistical significance was determined using a Log-rank test.


Figure 6 ( B ) E2F target genes expression was analyzed for correlation with expression of CASP2 , RAIDD and PIDD1 using Spearman ' s correlation coefficient . The central band represents the median , the boxes indicate the 2nd and 3rd quartile , and the whiskers are 1 . 5 times the interquartile range ( n = 200 genes ) . Correlation ( Pearson ) of CASP2 mRNA with transcript levels of Ki67 in the TCGA data set . Correlation ( Pearson ) of RAIDD and PIDD1 mRNA with transcript levels of Ki67 in the TCGA data set .

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

Figure 6(B) E2F target genes expression was analyzed for correlation with expression of CASP2, RAIDD and PIDD1 using Spearman's correlation coefficient. The central band represents the median, the boxes indicate the 2nd and 3rd quartile, and the whiskers are 1.5 times the interquartile range (n=200 genes).Correlation (Pearson) of CASP2 mRNA with transcript levels of Ki67 in the TCGA data set.Correlation (Pearson) of RAIDD and PIDD1 mRNA with transcript levels of Ki67 in the TCGA data set.


Figure 7 ( A ) The ploidy index is inferred as 1 / cell density of tumorous ( T ) and non - tumorous ( NT ) tissue of HCC patients with different liver diseases . ALD - alcoholic liver disease ( n = 65 ) ; HBV - hepatitis B virus infection ( n = 24 ) ; HCV - hepatitis C virus infection ( n = 85 ) ; NASH - non - alcoholic steatohepatitis ( n = 33 ) . Statistical significance was determined by one - way ANOVA with multiplicity correction ( Sidak - Holm ) ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . n - numbers refer to the number of patient samples analyzed . ( B ) All patients were grouped according to HCC tumor cell density as surrogate for tumor ploidy . Data were divided at the median and exact patient numbers are shown in the graphs . Median recurrence - free survival ( RFS ) for a period of 10 years for patients with low tumor ploidy was 4 . 8 years . For patients with high tumor ploidy the median RFS could not be defined as more than 50 % of the patients survived without relapse . Patient characteristics are shown in Appendix table S1 . A Log - rank test was used to determine statistical significance .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The ploidy index is inferred as 1/cell density of tumorous (T) and non-tumorous (NT) tissue of HCC patients with different liver diseases. ALD - alcoholic liver disease (n=65); HBV - hepatitis B virus infection (n=24); HCV - hepatitis C virus infection (n=85); NASH - non-alcoholic steatohepatitis (n=33). Statistical significance was determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01, *** p≤0.001. n-numbers refer to the number of patient samples analyzed.(B) All patients were grouped according to HCC tumor cell density as surrogate for tumor ploidy. Data were divided at the median and exact patient numbers are shown in the graphs. Median recurrence-free survival (RFS) for a period of 10 years for patients with low tumor ploidy was 4.8 years. For patients with high tumor ploidy the median RFS could not be defined as more than 50% of the patients survived without relapse. Patient characteristics are shown in Appendix table S1. A Log-rank test was used to determine statistical significance.


Figure 1A . Arginine methylase inhibitor decreases SIRT7 methylation . HEK293T cells expressing Flag - tagged SIRT7 were treated with increasing doses of AdOx as indicated for 24 hrs . After immunoprecipitated with Flag - beads , SIRT7 was analyzed by western blot and detected by an antibody against mono - methylarginine ( α - me1 ) . Relative methylation ratio ( Ratio ) was calculated by normalizing arginine methylation level against the Flag - SIRT7 protein . WCL , whole cell lysateC . PRMT inhibitor reduces arginine methylation of wildtype SIRT7 , but not its RK / RF mutants . HEK293T cells expressing wildtype SIRT7 or its RK / RF mutants were treated with or without AdOx for 24 hrs . Arginine methylation of immunopurified SIRT7 was determined by western blotD . SIRT7 is mono - and asymmetrically di - methylated at R388 . Flag - tagged SIRT7 and its RK / RF mutants were expressed in HEK293T cells . Immunopurified SIRT7 was blotted with antibodies against mono - methylarginine ( α - me1 ) , asymmetric di - methylarginine ( α - me2a ) , and symmetric di - methylarginine ( α - me2s ) , respectivelyE . R388 site - specific methylation antibody recognizes R388 - methylated peptide , but not the unmodified peptide . The unmodified peptide corresponding to R388 , R388 - monomethylated ( R388me1 ) peptide , R388 - asymmetrically di - methylated ( R388me2a ) peptide were blotted with α - meSIRT7 ( R388 ) antibodyF . R388 - methylated peptide , but not the unmodified peptide , blocks α - meSIRT7 ( R388 ) antibody . After incubation with unmodified peptide or R388 - methylated peptide ( R388me2a ) , α - meSIRT7 ( R388 ) antibody was used to detect immunopurified SIRT7G . RK and RF mutations disrupt SIRT7 methylation . Flag - tagged wildtype SIRT7 and its RK / RF mutants were immunoprecipitated and blotted with α - meSIRT7 ( R388 ) antibodyH . SIRT7 is methylated at R388 . HEK293T cells ectopically expressing Flag - tagged SIRT7 were treated with increasing concentrations of AdOx for 24 hrs as indicated . SIRT7 protein was pulled down by Flag - beads . Arginine methylation of SIRT7 was detected with a site - specific antibody against R388 methylation [ α - meSIRT7 ( R388 ) ] I - J . AdOx treatment decreases R388 methylation of SIRT7 . MEF and L02 cells were treated with AdOx for 24 hrs . Methylation of immunoprecipitated endogenous SIRT7 was determined by western blot ( I ) . The band intensity of western blots was measured to quantify relative R388 methylation ratio ( J ) ( mean ± SD , n = 3 experimental replicates , * * * p < 0 . 001 , unpaired two - tailed t - test ) K - L . Quantification of R388 - methylated endogenous SIRT7 in HEK293T and L02 cells . R388 - methylated endogenous SIRT7 was captured from HEK293T and L02 cells by using R388 site - specific methylation antibody ( K ) . Input , output , and immunoprecipitate were analyzed by western blot to quantify the percentage of R388 - methylated protein ( L ) ( mean ± SD , n = 3 experimental replicates )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Arginine methylase inhibitor decreases SIRT7 methylation. HEK293T cells expressing Flag-tagged SIRT7 were treated with increasing doses of AdOx as indicated for 24 hrs. After immunoprecipitated with Flag-beads, SIRT7 was analyzed by western blot and detected by an antibody against mono-methylarginine (α-me1). Relative methylation ratio (Ratio) was calculated by normalizing arginine methylation level against the Flag-SIRT7 protein. WCL, whole cell lysateC. PRMT inhibitor reduces arginine methylation of wildtype SIRT7, but not its RK/RF mutants. HEK293T cells expressing wildtype SIRT7 or its RK/RF mutants were treated with or without AdOx for 24 hrs. Arginine methylation of immunopurified SIRT7 was determined by western blotD. SIRT7 is mono- and asymmetrically di-methylated at R388. Flag-tagged SIRT7 and its RK/RF mutants were expressed in HEK293T cells. Immunopurified SIRT7 was blotted with antibodies against mono-methylarginine (α-me1), asymmetric di-methylarginine (α-me2a), and symmetric di-methylarginine (α-me2s), respectivelyE. R388 site-specific methylation antibody recognizes R388-methylated peptide, but not the unmodified peptide. The unmodified peptide corresponding to R388, R388-monomethylated (R388me1) peptide, R388-asymmetrically di-methylated (R388me2a) peptide were blotted with α-meSIRT7(R388) antibodyF. R388-methylated peptide, but not the unmodified peptide, blocks α-meSIRT7(R388) antibody. After incubation with unmodified peptide or R388-methylated peptide (R388me2a), α-meSIRT7(R388) antibody was used to detect immunopurified SIRT7G. RK and RF mutations disrupt SIRT7 methylation. Flag-tagged wildtype SIRT7 and its RK/RF mutants were immunoprecipitated and blotted with α-meSIRT7(R388) antibodyH. SIRT7 is methylated at R388. HEK293T cells ectopically expressing Flag-tagged SIRT7 were treated with increasing concentrations of AdOx for 24 hrs as indicated. SIRT7 protein was pulled down by Flag-beads. Arginine methylation of SIRT7 was detected with a site-specific antibody against R388 methylation [α-meSIRT7(R388)]I-J. AdOx treatment decreases R388 methylation of SIRT7. MEF and L02 cells were treated with AdOx for 24 hrs. Methylation of immunoprecipitated endogenous SIRT7 was determined by western blot (I). The band intensity of western blots was measured to quantify relative R388 methylation ratio (J) (mean ± SD, n=3 experimental replicates, ***p < 0.001, unpaired two-tailed t-test)K-L. Quantification of R388-methylated endogenous SIRT7 in HEK293T and L02 cells. R388-methylated endogenous SIRT7 was captured from HEK293T and L02 cells by using R388 site-specific methylation antibody (K). Input, output, and immunoprecipitate were analyzed by western blot to quantify the percentage of R388-methylated protein (L) (mean±SD, n=3 experimental replicates)


Figure 2A . SIRT7 binds to PRMT1 , PRMT3 , and PRMT6 . GFP - tagged PRMTs ( PRMT1 - 9 ) were expressed in HEK293T cells . PRMT protein was immunoprecipitated with GFP antibody and analyzed by western blotB . Co - expression of PRMT6 , but not PRMT1 or PRMT3 , increases R388 methylation of SIRT7 . Flag - tagged SIRT7 and GFP - tagged PRMT1 , PRMT3 , or PRMT6 were co - expressed in HEK293T cells . R388 methylation of immunopurified SIRT7 was analyzed by western blotC . Wildtype PRMT6 , but not its catalytic - deficient mutant , upregulates SIRT7 R388 methylation . Wildtype PRMT6 or its V86K / D88A mutant ( DM ) was co - expressed with Flag - tagged SIRT7 in HEK293T cells . R388 methylation of immunopurified SIRT7 was detected by western blotD - E . PRMT6 physically interacts with endogenous SIRT7 . Endogenous SIRT7 ( D ) or PRMT6 ( E ) was pulled down using corresponding antibody from HEK293T , MEF , and primary mouse hepatocytes , respectively . Input and immunoprecipitates were analyzed by western blotF . SIRT7 associates with PRMT6 in vitro . Recombinant GST - PRMT6 were incubated with HEK293T lysates . After GST pulldown , the interaction of recombinant protein with endogenous SIRT7 was analyzed by western blot . The arrows indicate GST and GST - PRMT6 protein , respectivelyG . PRMT6 inhibitor reduces arginine methylation of wildtype SIRT7 , but not RK / RF mutants . Wildtype SIRT7 or its RK / RF mutants were stably expressed in MEF cells . After treating cells with or without PRMT6 - specific chemical inhibitor ( PRMT6i ) for 24 hrs , SIRT7 was immunoprecipitated and blotted with R388 - specific methylation antibodyH . Prmt6 depletion decreases R388 methylation of endogenous Sirt7 . Scramble control cells ( scr ) or MEF cells stably expressing two different shRNAs ( # 1 and # 2 ) against Prmt6 was established . R388 methylation of immunopurified endogenous Sirt7 was determined by western blotI . Treatment with PRMT6 inhibitor reduces R388 methylation of endogenous SIRT7 in a dose - dependent manner . MEF and HEK293T cells were treated with increasing doses of PRMT6i for 24 hrs as indicated . R388 methylation of immunoprecipitated endogenous SIRT7 was determined by western blotJ - K . Prmt6 depletion reduces the percentage of R388 - methylated endogenous SIRT7 in MEF cells . R388 - methylated endogenous SIRT7 was captured from scramble control cells ( scr ) and Prmt6 - knockdown MEF cells by using R388 site - specific methylation antibody ( J ) . Input , output , and immunoprecipitate were analyzed by western blot to quantify the percentage of R388 - methylated protein ( K ) ( mean ± SD , n = 3 experimental replicates ) L . Recombinant PRMT6 directly methylates SIRT7 at R388 in vitro . Bacterially expressed PRMT6 and SIRT7 proteins were incubated with SAM or PRMT6i as indicated . Heat - inactivated PRMT6 was included as negative control R388 methylation of recombinant SIRT7 protein was determined by western

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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. SIRT7 binds to PRMT1, PRMT3, and PRMT6. GFP-tagged PRMTs (PRMT1-9) were expressed in HEK293T cells. PRMT protein was immunoprecipitated with GFP antibody and analyzed by western blotB. Co-expression of PRMT6, but not PRMT1 or PRMT3, increases R388 methylation of SIRT7. Flag-tagged SIRT7 and GFP-tagged PRMT1, PRMT3, or PRMT6 were co-expressed in HEK293T cells. R388 methylation of immunopurified SIRT7 was analyzed by western blotC. Wildtype PRMT6, but not its catalytic-deficient mutant, upregulates SIRT7 R388 methylation. Wildtype PRMT6 or its V86K/D88A mutant (DM) was co-expressed with Flag-tagged SIRT7 in HEK293T cells. R388 methylation of immunopurified SIRT7 was detected by western blotD-E. PRMT6 physically interacts with endogenous SIRT7. Endogenous SIRT7 (D) or PRMT6 (E) was pulled down using corresponding antibody from HEK293T, MEF, and primary mouse hepatocytes, respectively. Input and immunoprecipitates were analyzed by western blotF. SIRT7 associates with PRMT6 in vitro. Recombinant GST-PRMT6 were incubated with HEK293T lysates. After GST pulldown, the interaction of recombinant protein with endogenous SIRT7 was analyzed by western blot. The arrows indicate GST and GST-PRMT6 protein, respectivelyG. PRMT6 inhibitor reduces arginine methylation of wildtype SIRT7, but not RK/RF mutants. Wildtype SIRT7 or its RK/RF mutants were stably expressed in MEF cells. After treating cells with or without PRMT6-specific chemical inhibitor (PRMT6i) for 24 hrs, SIRT7 was immunoprecipitated and blotted with R388-specific methylation antibodyH. Prmt6 depletion decreases R388 methylation of endogenous Sirt7. Scramble control cells (scr) or MEF cells stably expressing two different shRNAs (#1 and #2) against Prmt6 was established. R388 methylation of immunopurified endogenous Sirt7 was determined by western blotI. Treatment with PRMT6 inhibitor reduces R388 methylation of endogenous SIRT7 in a dose-dependent manner. MEF and HEK293T cells were treated with increasing doses of PRMT6i for 24 hrs as indicated. R388 methylation of immunoprecipitated endogenous SIRT7 was determined by western blotJ-K. Prmt6 depletion reduces the percentage of R388-methylated endogenous SIRT7 in MEF cells. R388-methylated endogenous SIRT7 was captured from scramble control cells (scr) and Prmt6-knockdown MEF cells by using R388 site-specific methylation antibody (J). Input, output, and immunoprecipitate were analyzed by western blot to quantify the percentage of R388-methylated protein (K) (mean±SD, n=3 experimental replicates)L. Recombinant PRMT6 directly methylates SIRT7 at R388 in vitro. Bacterially expressed PRMT6 and SIRT7 proteins were incubated with SAM or PRMT6i as indicated. Heat-inactivated PRMT6 was included as negative control R388 methylation of recombinant SIRT7 protein was determined by western blot


Figure 3A . RF , but not RK mutant , suppresses H3K18 deacetylase activity of SIRT7 . Flag - SIRT7 and its RK / RF mutants were expressed in HEK293T and MEF cells , respectively . Cell lysates were subjected to western blot to detect histone H3K18 acetylation . H3K27ac and H3K56ac were included as negative control . The level of each histone acetylation marker was normalized against histone H3 ( Ratio ) B . R388 methylation of SIRT7 decreases its H3K18 deacetylase activity . HEK293T cells expressing wildtype SIRT7 or its RK / RF mutants were treated with or without AdOx for 24 hrs . The SIRT7 enzyme was immunopurified with Flag - beads and further incubated with extracted chromatin in vitro . R388 methylation of immunopurified SIRT7 and H3K18ac of in vitro deacetylated chromatin were determined by western blotC . RF , but not RK mutant , shows reduced H3K18 deacetylase activity in vitro . Chromatin was extracted from HEK293T cells and incubated with recombinant GST - tagged SIRT7 ( wildtype and RK / RF mutants ) in vitro for 2 hrs . H3K18ac was determined by western blotD . Wildtype PRMT6 , but not its methylase - dead mutant , decreases the H3K18 deacetylase activity of SIRT7 . Flag - tagged SIRT7 was co - expressed with wildtype PRMT6 or its catalytic - inactive mutant ( DM ) in HEK293T cells . The H3K18 deacetylase activity of immunopurified SIRT7 was assayed in vitroE . PRMT6 deficiency promotes the H3K18 deacetylase activity of SIRT7 . Control sgRNA or two independent sgRNAs ( # 1 and # 2 ) against PRMT6 was introduced into HEK293T cells . Flag - tagged SIRT7 was ectopically expressed in these cells . The histone deacetylase activity of immunoprecipitated SIRT7 was assayed in vitro using chromatin as a substrate . R388 methylation of immunopurified SIRT7 and H3K18 acetylation level of in vitro deacetylated chromatin was determined by western blotF - G . Methylated recombinant SIRT7 shows reduced H3K18 deacetylase activity . In vitro methylated SIRT7 from Fig 2L was subjected to in vitro deacetylation assay . H3K18 acetylation level of in vitro deacetylated chromatin was determined by western blot ( F ) . Relative H3K18ac level was quantified from three independent experiments ( G ) ( mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 , n . s . = not significant , unpaired two - tailed t test )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RF, but not RK mutant, suppresses H3K18 deacetylase activity of SIRT7. Flag-SIRT7 and its RK/RF mutants were expressed in HEK293T and MEF cells, respectively. Cell lysates were subjected to western blot to detect histone H3K18 acetylation. H3K27ac and H3K56ac were included as negative control. The level of each histone acetylation marker was normalized against histone H3 (Ratio)B. R388 methylation of SIRT7 decreases its H3K18 deacetylase activity. HEK293T cells expressing wildtype SIRT7 or its RK/RF mutants were treated with or without AdOx for 24 hrs. The SIRT7 enzyme was immunopurified with Flag-beads and further incubated with extracted chromatin in vitro. R388 methylation of immunopurified SIRT7 and H3K18ac of in vitro deacetylated chromatin were determined by western blotC. RF, but not RK mutant, shows reduced H3K18 deacetylase activity in vitro. Chromatin was extracted from HEK293T cells and incubated with recombinant GST-tagged SIRT7 (wildtype and RK/RF mutants) in vitro for 2 hrs. H3K18ac was determined by western blotD. Wildtype PRMT6, but not its methylase-dead mutant, decreases the H3K18 deacetylase activity of SIRT7. Flag-tagged SIRT7 was co-expressed with wildtype PRMT6 or its catalytic-inactive mutant (DM) in HEK293T cells. The H3K18 deacetylase activity of immunopurified SIRT7 was assayed in vitroE. PRMT6 deficiency promotes the H3K18 deacetylase activity of SIRT7. Control sgRNA or two independent sgRNAs (#1 and #2) against PRMT6 was introduced into HEK293T cells. Flag-tagged SIRT7 was ectopically expressed in these cells. The histone deacetylase activity of immunoprecipitated SIRT7 was assayed in vitro using chromatin as a substrate. R388 methylation of immunopurified SIRT7 and H3K18 acetylation level of in vitro deacetylated chromatin was determined by western blotF-G. Methylated recombinant SIRT7 shows reduced H3K18 deacetylase activity. In vitro methylated SIRT7 from Fig 2L was subjected to in vitro deacetylation assay. H3K18 acetylation level of in vitro deacetylated chromatin was determined by western blot (F). Relative H3K18ac level was quantified from three independent experiments (G) (mean±SD, n=3 experimental replicates, **p<0.01, n.s.=not significant, unpaired two-tailed t test)


Figure 4Wildtype SIRT7 or its RF mutant was re - expressed in Sirt7 - knockdown MEF cells ( A ) Wildtype SIRT7 , but not its RF mutant , restores mitochondria mas in Sirt7 - deficient MEF cells Mitochondria mass was determined by Mito Tracker Red staining ( B ) ( mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; n . s . = not significant , unpaired two - tailed t test ) Wildtype SIRT7 , but not its RF mutant , restore ATP leve in Sirt7 - deficient MEF cells Cellular ATP was quantified and normalized to cell number ( C ) ( mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; n . s . = not significant , unpaired two - tailed t test ) Wildtype SIRT7 , but not its RF mutant , restore cellular respiration in Sirt7 - deficient MEF cells Respiration flux was determined as indicated ( D ) . ( mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; n . s . = not significant unpaired two - tailed t test ) Wildtype SIRT7 , but not its RF mutant , downregulates SIRT7 target genes involved in mitochondria biogenesis . Wildtype SIRT7 and its RF mutant were stably expressed in MEF cells ( E ) Wildtype SIRT7 , but not its RF mutant , downregulates SIRT7 target genes involved in mitochondria biogenesis SIRT7 target gene expression was determined and presented as a heatmap on a log2 scale ( F ) Control cells and stable Sirt7 - knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs . The H3K18ac level at the promoter of SIRT7 target genes ( Gfm2 , Mrps31 , Mrps33 , and Mrpl40 ) was determined by ChIP - qPCR , Rabbit IgG was used as a negative control ( G ) ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - tailed t testInhibition of Prmt6 decreases mitochondria mas of control cells , but not Sirt7 - knockdown MEF cells Control cells and stable Sirt7 - knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Mitochondria mass was determined by Mito Tracker Red staining ( H ) ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - tailed t testInhibition of Prmt6 decrease ATP leve of control cells , but not Sirt7 - knockdown MEF cells . Control cells and stable Sirt7 - knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Cellular ATP was quantified and normalized to cell number ( I ) ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - tailed t testInhibition of Prmt6 decrease the respiration flux of control cells , but not Sirt7 - knockdown MEF cells . Control cells and stable Sirt7 - knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Respiration flux was determined as indicated ( J ) . ( Data shown are mean ± SD , n = 3 experimental replicates , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; n . s . = not significant , unpaired two - tailed t

O B-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Wildtype SIRT7 or its RF mutant was re-expressed in Sirt7-knockdown MEF cells (A)Wildtype SIRT7, but not its RF mutant, restores mitochondria mas in Sirt7-deficient MEF cells Mitochondria mass was determined by Mito Tracker Red staining (B) (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant, unpaired two-tailed t test)Wildtype SIRT7, but not its RF mutant, restore ATP leve in Sirt7-deficient MEF cells Cellular ATP was quantified and normalized to cell number (C) (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant, unpaired two-tailed t test)Wildtype SIRT7, but not its RF mutant, restore cellular respiration in Sirt7-deficient MEF cells Respiration flux was determined as indicated (D). (mean ± SD, n=3 experimental replicates, *p<0.05; **p<0.01; n.s.=not significant unpaired two-tailed t test)Wildtype SIRT7, but not its RF mutant, downregulates SIRT7 target genes involved in mitochondria biogenesis. Wildtype SIRT7 and its RF mutant were stably expressed in MEF cells (E)Wildtype SIRT7, but not its RF mutant, downregulates SIRT7 target genes involved in mitochondria biogenesis SIRT7 target gene expression was determined and presented as a heatmap on a log2 scale (F)Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs. The H3K18ac level at the promoter of SIRT7 target genes (Gfm2, Mrps31, Mrps33, and Mrpl40) was determined by ChIP-qPCR, Rabbit IgG was used as a negative control (G) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t testInhibition of Prmt6 decreases mitochondria mas of control cells, but not Sirt7-knockdown MEF cells Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Mitochondria mass was determined by Mito Tracker Red staining (H) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t testInhibition of Prmt6 decrease ATP leve of control cells, but not Sirt7-knockdown MEF cells. Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Cellular ATP was quantified and normalized to cell number (I) ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t testInhibition of Prmt6 decrease the respiration flux of control cells, but not Sirt7-knockdown MEF cells. Control cells and stable Sirt7-knockdown MEF cells were treated with or without 5 µM PRMT6i for 24 hrs Respiration flux was determined as indicated (J). ( Data shown are mean±SD, n=3 experimental replicates, *p<0.05; **p<0.01; ***p < 0.001; n.s.=not significant, unpaired two-tailed t test


Figure 5A . Glucose depletion reduces SIRT7 methylation . MEF cells stably expressing Flag - SIRT7 were cultured in medium depleted with glucose ( 12 hrs ) , FBS ( 12 hrs ) or glutamine ( 24 hrs ) . R388 methylation of immunopurified SIRT7 and Ampk phosphorylation were determined by western blot . R388 methylation level was normalized to Flag - SIRT7 ( Ratio ) B . Glucose starvation decreases R388 methylation of SIRT7 in a time - dependent manner . MEF cells stably expressing Flag - SIRT7 were cultured in glucose - free media as indicated . R388 methylation level of immunopurified SIRT7 was determined by western blotC . Glucose dose - dependently upregulates R388 methylation of SIRT7 . MEF cells stably expressing Flag - SIRT7 were cultured with increasing concentrations of glucose as indicated for 12 hrs . R388 methylation of SIRT7 and Ampk phosphorylation were determined by western blotD . AICAR treatment decreases R388 methylation of SIRT7 . MEF cells stably expressing Flag - SIRT7 were treated with or without AICAR for 12 hrs . R388 methylation of immunoprecipitated SIRT7 was determined by western blotE - F . AICAR reduces the percentage of R388 - methylated endogenous SIRT7 in MEF cells . R388 - methylated endogenous SIRT7 was captured from control and AICAR - treated MEF cells by using R388 site - specific methylation antibody ( E ) . Input , output , and immunoprecipitate were analyzed by western blot to quantify the percentage of R388 - methylated protein ( F ) ( mean ± SD , n = 3 experimental replicates ) G . AMPK deletion increases SIRT7 R388 methylation . Flag - tagged SIRT7 was stably expressed in Ampk wildtype ( WT ) and Ampk α1 / α2 double - knockout ( DKO ) MEF cells and 293A cells . SIRT7 protein was immunopurified with Flag - beads , R388 methylation was determined by western blotH . AMPK is required for glucose starvation - induced R388 hypomethylation of SIRT7 . Ampk wildtype ( WT ) and Ampk α1 / α2 double - knockout ( DKO ) MEF cells stably expressing Flag - SIRT7 were cultured with or without glucose for 12 hrs . SIRT7 was purified with Flag - beads and R388 methylation was determined by western blotI . PRMT6 is necessary for AMPK - induced SIRT7 hypomethylation . Flag - tagged SIRT7 was stably expressed in scramble control MEF cells or cells expressing two independent shRNAs targeting Prmt6 ( # 1 and # 2 ) . Cells were cultured with or without glucose for 12 hrs . R388 methylation of immunopurified SIRT7 was determined by western blotJ . PRMT6 is necessary for AMPK - induced SIRT7 hypomethylation . Wildtype MEFs and Ampk DKO cells were treated with PRMT6i for 24 hrs . SIRT7 was purified by Flag - beads , and R388 methylation of SIRT7 was determined by western blotK . Both AICAR treatment and glucose depletion impair the association between Prmt6 and Sirt7 . MEF cells were cultured with or without glucose and treated with AICAR as indicated . Co - immunoprecipitation was performed using SIRT7 antibody . Input and immunoprecipitates were analyzed by western blotL . R388 methylation of SIRT7 does not modulate its phosphorylation . MEF cells stably expressing Flag - tagged wildtype SIRT7 or RK mutant were treated with PRMT6i ( 24 hrs ) and cultured with or without glucose ( 12 hrs ) as indicated . SIRT7 protein was immunopurified with Flag - beads , R388 methylation and phosphorylation of SIRT7 were determined by western

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Glucose depletion reduces SIRT7 methylation. MEF cells stably expressing Flag-SIRT7 were cultured in medium depleted with glucose (12 hrs), FBS (12 hrs) or glutamine (24 hrs). R388 methylation of immunopurified SIRT7 and Ampk phosphorylation were determined by western blot. R388 methylation level was normalized to Flag-SIRT7 (Ratio)B. Glucose starvation decreases R388 methylation of SIRT7 in a time-dependent manner. MEF cells stably expressing Flag-SIRT7 were cultured in glucose-free media as indicated. R388 methylation level of immunopurified SIRT7 was determined by western blotC. Glucose dose-dependently upregulates R388 methylation of SIRT7. MEF cells stably expressing Flag-SIRT7 were cultured with increasing concentrations of glucose as indicated for 12 hrs. R388 methylation of SIRT7 and Ampk phosphorylation were determined by western blotD. AICAR treatment decreases R388 methylation of SIRT7. MEF cells stably expressing Flag-SIRT7 were treated with or without AICAR for 12 hrs. R388 methylation of immunoprecipitated SIRT7 was determined by western blotE-F. AICAR reduces the percentage of R388-methylated endogenous SIRT7 in MEF cells. R388-methylated endogenous SIRT7 was captured from control and AICAR-treated MEF cells by using R388 site-specific methylation antibody (E). Input, output, and immunoprecipitate were analyzed by western blot to quantify the percentage of R388-methylated protein (F) (mean±SD, n=3 experimental replicates)G. AMPK deletion increases SIRT7 R388 methylation. Flag-tagged SIRT7 was stably expressed in Ampk wildtype (WT) and Ampk α1/α2 double-knockout (DKO) MEF cells and 293A cells. SIRT7 protein was immunopurified with Flag-beads, R388 methylation was determined by western blotH. AMPK is required for glucose starvation-induced R388 hypomethylation of SIRT7. Ampk wildtype (WT) and Ampk α1/α2 double-knockout (DKO) MEF cells stably expressing Flag-SIRT7 were cultured with or without glucose for 12 hrs. SIRT7 was purified with Flag-beads and R388 methylation was determined by western blotI. PRMT6 is necessary for AMPK-induced SIRT7 hypomethylation. Flag-tagged SIRT7 was stably expressed in scramble control MEF cells or cells expressing two independent shRNAs targeting Prmt6 (#1 and #2). Cells were cultured with or without glucose for 12 hrs. R388 methylation of immunopurified SIRT7 was determined by western blotJ. PRMT6 is necessary for AMPK-induced SIRT7 hypomethylation. Wildtype MEFs and Ampk DKO cells were treated with PRMT6i for 24 hrs. SIRT7 was purified by Flag-beads, and R388 methylation of SIRT7 was determined by western blotK. Both AICAR treatment and glucose depletion impair the association between Prmt6 and Sirt7. MEF cells were cultured with or without glucose and treated with AICAR as indicated. Co-immunoprecipitation was performed using SIRT7 antibody. Input and immunoprecipitates were analyzed by western blotL. R388 methylation of SIRT7 does not modulate its phosphorylation. MEF cells stably expressing Flag-tagged wildtype SIRT7 or RK mutant were treated with PRMT6i (24 hrs) and cultured with or without glucose (12 hrs) as indicated. SIRT7 protein was immunopurified with Flag-beads, R388 methylation and phosphorylation of SIRT7 were determined by western blot


Figure 6Glucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte . After fasting for 16 hrs , mice were injected with glucose . Hepatic tissue was harvested in a time - dependent manner as indicated ( n = 3 for each time point ) . SIRT7 protein was immunoprecipitated from whole liver extracts . R388 methylation of Sirt7 was determined by western blot ( A Scale bar in the upper panel , 500 µm ; scale bar in the lower panel , 100 µmGlucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte . After fasting for 16 hrs , mice were injected with glucose . Hepatic tissue was harvested in a time - dependent manner as indicated ( n = 3 for each time point ) . SIRT7 protein was immunoprecipitated from whole liver extracts . R388 methylation of Sirt7 was determined b immunohistochemical staining ( B ) . Scale bar in the upper panel , 500 µm ; scale bar in the lower panel , 100 µmAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7 - target genes . MEF cells stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs . R388 methylation of SIRT7 was determined by western blot ( C ) ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7 - target genes . MEF cells stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs H3K18ac level at SIRT7 - target gene promoters were determined by ChIP - qPCR . Normal rabbit IgG was used as a negative control ( D ) ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7 - target genes . MEF cells stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs mRNA expression was determined by qPCR analysis ( E ) ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7 - target genes . MEF cells stably expressing Flag - SIRT7 were incubated with or without PRMT6i for 12 hrs , followed by treatment with 1 mM AICAR for 12 hrs Mitochondria mass was determined by Mito Tracker Red staining ( F ) . ( Data shown are mean ± SD , n = 3 experimental replicates , * * p < 0 . 01 ; * * * p < 0 . 001 , unpaired two - tailed t

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6Glucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte. After fasting for 16 hrs, mice were injected with glucose. Hepatic tissue was harvested in a time-dependent manner as indicated (n=3 for each time point). SIRT7 protein was immunoprecipitated from whole liver extracts. R388 methylation of Sirt7 was determined by western blot (A Scale bar in the upper panel, 500 µm; scale bar in the lower panel, 100 µmGlucose administration leads to R388 hypermethylation of Sirt7 in mouse hepatocyte. After fasting for 16 hrs, mice were injected with glucose. Hepatic tissue was harvested in a time-dependent manner as indicated (n=3 for each time point). SIRT7 protein was immunoprecipitated from whole liver extracts. R388 methylation of Sirt7 was determined b immunohistochemical staining (B). Scale bar in the upper panel, 500 µm; scale bar in the lower panel, 100 µmAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs. R388 methylation of SIRT7 was determined by western blot (C) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs H3K18ac level at SIRT7-target gene promoters were determined by ChIP-qPCR. Normal rabbit IgG was used as a negative control (D) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs mRNA expression was determined by qPCR analysis (E) (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t testAICAR and PRMT6 inhibitor decrease R388 methylation and epigenetically downregulate SIRT7-target genes. MEF cells stably expressing Flag-SIRT7 were incubated with or without PRMT6i for 12 hrs, followed by treatment with 1 mM AICAR for 12 hrs Mitochondria mass was determined by Mito Tracker Red staining (F). (Data shown are mean±SD, n=3 experimental replicates, **p<0.01; ***p < 0.001, unpaired two-tailed t test


Figure 1B , C . Detection of CST at stalled and active forks with SIRF assay in U2OS ( B ) and HCT116 ( C ) cells . To detect CST at stalled forks , cells were pulse - labeled with EdU for 8 min , followed by 4 mM HU treatment for 3 h . To detect CST at active forks , SIRF was performed without HU treatment . Representative SIRF images of CTC1 , STN1 and PCNA on normal or stalled forks are shown . Scale bars : 10 µm . Relative SIRF fluorescence intensity was calculated by normalizing to untreated samples and then to their respective EdU - SIRF signals . Mean value in each sample is labeled in the graph . N : the number of cells analyzed in each sample .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1B, C. Detection of CST at stalled and active forks with SIRF assay in U2OS (B) and HCT116 (C) cells. To detect CST at stalled forks, cells were pulse-labeled with EdU for 8 min, followed by 4 mM HU treatment for 3 h. To detect CST at active forks, SIRF was performed without HU treatment. Representative SIRF images of CTC1, STN1 and PCNA on normal or stalled forks are shown. Scale bars: 10 µm. Relative SIRF fluorescence intensity was calculated by normalizing to untreated samples and then to their respective EdU-SIRF signals. Mean value in each sample is labeled in the graph. N: the number of cells analyzed in each sample.


Figure 2Western blot showing STN1 knockdown . shLUC ( siRNA targeting luciferase ) was used as the control . CST deficiency causes ssDNA accumulation upon fork stalling . HeLa cells with STN1 knockdown were incubated with 10 µM BrdU for 48 h , followed by 2 mM HU treatment for 3 h , and subsequently stained with BrdU antibody under a non - denaturing condition to detect ssDNA . N , the number of cells analyzed in each condition . Relative BrdU fluorescence intensity was quantitated by Image J . Scheme of DNA fiber analysis and representative fiber images . DNA fiber analysis of U2OS cells with STN1 knockdown and its RNAi - resistant Flag - STN1 WT re - expression with and without mirin treatment . DNA fiber analysis of BJ cells with STN1 knockdown by two siRNA sequence with and without mirin treatment . DNA fiber analysis of HCT116 cells with STN1 knockdown with and without mirin treatment . DNA fiber analysis of U2OS cells showing that SMARCAL1 - mediated fork reversal is needed for nascent - stand degradation observed in CST - deficient cells . DNA fiber analysis of U2OS cells showing that ZRANB3 - mediated fork reversal is needed for nascent - strand degradation observed in CST - deficient cells .

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Figure 2Western blot showing STN1 knockdown. shLUC (siRNA targeting luciferase) was used as the control.CST deficiency causes ssDNA accumulation upon fork stalling. HeLa cells with STN1 knockdown were incubated with 10 µM BrdU for 48 h, followed by 2 mM HU treatment for 3 h, and subsequently stained with BrdU antibody under a non-denaturing condition to detect ssDNA. N, the number of cells analyzed in each condition. Relative BrdU fluorescence intensity was quantitated by Image J.Scheme of DNA fiber analysis and representative fiber images.DNA fiber analysis of U2OS cells with STN1 knockdown and its RNAi-resistant Flag-STN1 WT re-expression with and without mirin treatment.DNA fiber analysis of BJ cells with STN1 knockdown by two siRNA sequence with and without mirin treatment.DNA fiber analysis of HCT116 cells with STN1 knockdown with and without mirin treatment.DNA fiber analysis of U2OS cells showing that SMARCAL1-mediated fork reversal is needed for nascent-stand degradation observed in CST-deficient cells.DNA fiber analysis of U2OS cells showing that ZRANB3-mediated fork reversal is needed for nascent-strand degradation observed in CST-deficient cells.


Figure 3SIRF detection of MRE11 at stalled forks in STN1 - deficient U2OS cells . Representative SIRF images of MRE11 at normal or stalled replication forks are shown . Scale bars : 10 µm . ssDNA accumulation analysis in STN1 - deficient U2OS cells with and without mirin treatment . Representative images of native BrdU staining are shown .

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Figure 3SIRF detection of MRE11 at stalled forks in STN1-deficient U2OS cells. Representative SIRF images of MRE11 at normal or stalled replication forks are shown. Scale bars: 10 µm.ssDNA accumulation analysis in STN1-deficient U2OS cells with and without mirin treatment. Representative images of native BrdU staining are shown.


Figure 4Coomassie blue stained SDS - PAGE gel ( 15 % ) of purified human MRE11 . ( i ) Scheme shows the nuclease activity of MRE11 in degrading 5 ' Cy3 - labeled substrates ( 25 nt + 60 nt with phosphorothioate bonds on both ends ) . ( ii ) The 5 ' Cy3 - labeled substrates ( 80 nM ) were incubated with the indicated concentration of MRE11 with or without the Mn2 + cofactor at 37 ° C for 20 min . Reactions were stopped by SDS and proteinase K and resolved on 27 % denatured polyacrylamide gel . ( iii ) The results are graphed , and error bars represent the standard deviation ( ± SD ) calculated from at least three independent experiments . Coomassie blue stained SDS - PAGE gel ( 15 % ) of purified human CST complex ( CTC1 wild - type and ∆ 700N ) . DNA binding activity of wild - type CST complex and Δ700N mutant complex . The 5 ' Cy3 - labeled substrates ( 80 nM ) were incubated with the indicated concentrations of CST . Samples were analyzed by 0 . 8 % agarose gel . MRE11 degradation analysis . 5 ' Cy3 - labeled substrates ( 80 nM ) were pre - incubated with indicated concentrations of CST wild - type ( i ) or ∆ 700N mutant variant ( ii ) at 37 ° C for 5 min . Reactions were completed by adding MRE11 ( 200 nM ) for an additional 20 min incubation and then stopped by SDS and proteinase K . Samples were resolved in 27 % denatured polyacrylamide gel . The results are graphed and error bars represent the standard deviation ( ± SD ) calculated from at least three independent experiments .

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Figure 4Coomassie blue stained SDS-PAGE gel (15%) of purified human MRE11.(i) Scheme shows the nuclease activity of MRE11 in degrading 5' Cy3-labeled substrates (25 nt + 60 nt with phosphorothioate bonds on both ends). (ii) The 5' Cy3-labeled substrates (80 nM) were incubated with the indicated concentration of MRE11 with or without the Mn2+ cofactor at 37°C for 20 min. Reactions were stopped by SDS and proteinase K and resolved on 27% denatured polyacrylamide gel. (iii) The results are graphed, and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.Coomassie blue stained SDS-PAGE gel (15%) of purified human CST complex (CTC1 wild- type and ∆700N).DNA binding activity of wild-type CST complex and Δ700N mutant complex. The 5' Cy3- labeled substrates (80 nM) were incubated with the indicated concentrations of CST. Samples were analyzed by 0.8 % agarose gel.MRE11 degradation analysis. 5' Cy3-labeled substrates (80 nM) were pre-incubated with indicated concentrations of CST wild-type (i) or ∆700N mutant variant (ii) at 37°C for 5 min. Reactions were completed by adding MRE11 (200 nM) for an additional 20 min incubation and then stopped by SDS and proteinase K. Samples were resolved in 27% denatured polyacrylamide gel. The results are graphed and error bars represent the standard deviation (± SD) calculated from at least three independent experiments.


Figure 5Deletion of the N - terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres . U2OS stably expressing vector ( V ) , WT Myc - CTC1 and Myc - ∆ 700N were treated with or without HU and co - stained with Myc ( red ) and TRF2 ( green ) antibodies . Boxed areas are amplified in inserts to indicate CTC1 / TRF2 colocalization ( yellow ) . Scale bars : 10 μm . Quantification of percent of cells with > 3 CTC1 / TRF2 colocalization foci were from three independent experiments . In each experiment , > 150 cells from each sample were analyzed . ∆ 700N does not affect CST complex formation . HEK293T cells were co - transfected with Myc - CTC1 or Myc - ∆ 700N , His6 - STN1 and HA - TEN1 . Co - IP was performed with Myc antibody to pulldown His6 - STN1 and HA - TEN1 . Full - length Myc - CTC1 was prone to degradation during immunoprecipitation . ∆ 700N retains RAD51 interaction . HEK293T cells were co - transfected with Myc - CTC1 or Myc - ∆ 700N , His6 - STN1 , HA - TEN1 and Flag - RAD51 or vector control ( V ) , and treated with HU ( 2 mM , 16 h ) . Co - IP was performed with Flag antibody to pulldown Myc - CTC1 . DNA fiber analysis of CTC1 depleted U2OS cells with RNAi - resistant WT or ∆ 700N re - expression . Mean values in each sample are listed at the top of the graph . N , the number of cells analyzed in each condition , * * *

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Figure 5Deletion of the N-terminal 700 aa of CTC1 abolishes CTC1 localization at telomeres. U2OS stably expressing vector (V), WT Myc-CTC1 and Myc-∆700N were treated with or without HU and co-stained with Myc (red) and TRF2 (green) antibodies. Boxed areas are amplified in inserts to indicate CTC1/TRF2 colocalization (yellow).Scale bars: 10 μm. Quantification of percent of cells with >3 CTC1/TRF2 colocalization foci were from three independent experiments. In each experiment, >150 cells from each sample were analyzed.   ∆700N does not affect CST complex formation. HEK293T cells were co-transfected with Myc- CTC1 or Myc-∆700N, His6-STN1 and HA-TEN1. Co-IP was performed with Myc antibody to pulldown His6- STN1 and HA-TEN1. Full-length Myc-CTC1 was prone to degradation during immunoprecipitation.      ∆700N retains RAD51 interaction. HEK293T cells were co-transfected with Myc-CTC1 or Myc-∆700N, His6- STN1, HA-TEN1 and Flag-RAD51 or vector control (V), and treated with HU (2 mM, 16 h). Co-IP was performed with Flag antibody to pulldown Myc-CTC1.   DNA fiber analysis of CTC1 depleted U2OS cells with RNAi-resistant WT or ∆700N re-expression. Mean values in each sample are listed at the top of the graph. N, the number of cells analyzed in each condition, ***


Figure 6Micronuclei ( MN ) formation in BRCA2 - and STN1 - deficient cells . Co - depletion was achieved by transfecting siBRCA2 in U2OS cells expressing shSTN1 . Representative DAPI staining images are shown . Arrows point to MN . Scale bars : 20 µm . Average percentages of nuclei containing MN from three independent experiments are presented . In each experiment , > 300 nuclei were analyzed per sample . Anaphase bridges ( arrows ) in BRCA2 - and STN1 - deficient U2OS cells . Scale bar : 10 µm . Average percentages of anaphase bridges from three independent experiments are presented . γH2AX induced by BRCA2 knockdown and STN1 knockdown in U2OS cells . Nuclei containing ≥ 5 foci were considered as positive γ - H2AX staining . Results were from three independent knockdown experiments . In each experiment , > 80 cells were analyzed per sample . Co - depletion of STN1 and BRCA2 significantly impairs DNA replication . Scale bar : 50 µm . Results were from three independent knockdown experiments . In each experiment , > 180 nuclei were analyzed per sample . Co - depletion of STN1 and BRCA2 increases chromosome instabilities . U2OS cells with siBRCA2 and / or shSTN1 knockdown were treated with HU ( 2 mM , 3 h ) . Representative metaphase images show aberrant chromosomes ( red arrows ) . Boxed areas are amplified and shown at the bottom of images . Scale bars : 20 µm . Two independent knockdown and chromosome spread experiments were performed . N , the number of metaphase spreads analyzed in each sample .

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Figure 6Micronuclei (MN) formation in BRCA2- and STN1-deficient cells. Co-depletion was achieved by transfecting siBRCA2 in U2OS cells expressing shSTN1. Representative DAPI staining images are shown. Arrows point to MN. Scale bars: 20 µm. Average percentages of nuclei containing MN from three independent experiments are presented. In each experiment, >300 nuclei were analyzed per sample.Anaphase bridges (arrows) in BRCA2- and STN1-deficient U2OS cells. Scale bar: 10 µm. Average percentages of anaphase bridges from three independent experiments are presented.   γH2AX induced by BRCA2 knockdown and STN1 knockdown in U2OS cells. Nuclei containing ≥ 5 foci were considered as positive γ-H2AX staining. Results were from three independent knockdown experiments. In each experiment, >80 cells were analyzed per sample.   Co-depletion of STN1 and BRCA2 significantly impairs DNA replication. Scale bar: 50 µm. Results were from three independent knockdown experiments. In each experiment, >180 nuclei were analyzed per sample.Co-depletion of STN1 and BRCA2 increases chromosome instabilities. U2OS cells with siBRCA2 and/or shSTN1 knockdown were treated with HU (2 mM, 3 h). Representative metaphase images show aberrant chromosomes (red arrows). Boxed areas are amplified and shown at the bottom of images. Scale bars: 20 µm. Two independent knockdown and chromosome spread experiments were performed. N, the number of metaphase spreads analyzed in each sample.


Figure 7SIRF analysis of RAD51 binding to forks . Representative RAD51 SIRF images at normal or stalled forks are shown . Scale bars : 10 µm . Two independent treatments were performed and scatter plot of RAD51 - bound replication sites from one experiment is shown . Mean values in each sample are listed at the top of the graph . N , number of cells analyzed in each sample in one experiment . RAD51 foci formation in U2OS CTC1 knockdown cells was rescued by RNAi - resistant WT but not by ∆ 700N . Cells were treated with HU ( 2 mM , 16 h ) and stained with anti - RAD51 . Representative images are shown . Scale bars : 10 µm . Scatter plot of RAD51 foci number with and without HU treatment is shown . Mean values in each condition are listed at the top of the graph . DNA fiber analysis of nascent - strand DNA degradation in U2OS cells with STN1 and RAD51 co - depletion . Three independent treatments were performed and results from one biological replicate are shown . N , the number of DNA fibers analyzed in each sample in one experiment . Western blots showing protein knockdown in DNA fiber assays are included .

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Figure 7SIRF analysis of RAD51 binding to forks. Representative RAD51 SIRF images at normal or stalled forks are shown. Scale bars: 10 µm. Two independent treatments were performed and scatter plot of RAD51-bound replication sites from one experiment is shown. Mean values in each sample are listed at the top of the graph. N, number of cells analyzed in each sample in one experiment.RAD51 foci formation in U2OS CTC1 knockdown cells was rescued by RNAi-resistant WT but not by ∆700N. Cells were treated with HU (2 mM, 16 h) and stained with anti-RAD51. Representative images are shown. Scale bars: 10 µm. Scatter plot of RAD51 foci number with and without HU treatment is shown. Mean values in each condition are listed at the top of the graph.DNA fiber analysis of nascent-strand DNA degradation in U2OS cells with STN1 and RAD51 co-depletion. Three independent treatments were performed and results from one biological replicate are shown. N, the number of DNA fibers analyzed in each sample in one experiment. Western blots showing protein knockdown in DNA fiber assays are included.


Figure 1 ( B - D ) Kaplan - Meier survival plots comparing the survival probability of all melanoma patients ( B ) , BRAF - mutated patients ( C ) and BRAF wild - type ( WT ) patients ( D ) with high - ( magenta ) and low ( black ) MCUA mRNA expression . ( E - G ) Kaplan - Meier survival plots comparing the survival probability of patients of indicated cancer types that are divided into either high - ( magenta ) or low ( black ) MCUA mRNA expression . P - values were calculated by using a log - rank test . Abbreviations : KICH = kidney chromophobe ; KIRC = kidney renal clear cell carcinoma ; KIRP = kidney renal papillary cell carcinoma ; LUAD = lung adenocarcinoma ; LUSC = lung squamous cell carcinoma ; PAAD = pancreatic adenocarcinoma .

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Figure 1(B-D) Kaplan-Meier survival plots comparing the survival probability of all melanoma patients (B), BRAF-mutated patients (C) and BRAF wild-type (WT) patients (D) with high- (magenta) and low (black) MCUA mRNA expression.(E-G) Kaplan-Meier survival plots comparing the survival probability of patients of indicated cancer types that are divided into either high- (magenta) or low (black) MCUA mRNA expression. P-values were calculated by using a log-rank test. Abbreviations: KICH = kidney chromophobe; KIRC = kidney renal clear cell carcinoma; KIRP = kidney renal papillary cell carcinoma; LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; PAAD = pancreatic adenocarcinoma.


Figure 3 ( C ) Representative images of the migrated stained 1205Lu shCTRL ( black frame ) , shMCU _ 1 ( darker blue frame ) and shMCU _ 2 ( lighter blue frame ) cells on the lower side of the insert . ( D ) Quantification of the transwell migration in 1205Lu stable MCUA _ KD lines , based on the number of stained cells ( n = 4 biological replicates / condition , shown also by individual data points ) . Data information : Data are presented as mean ± SEM . Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test ( shMCU cells were compared to their respective control , shCTRL ) , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) . ( E ) Representative images of 1205Lu shCTRL ( black frame ) , shMCU _ 1 ( darker blue frame ) and shMCU _ 2 ( lighter blue frame ) melanoma spheroids after 72 h invasion in collagen . Live cells are shown in green . Scale bar : 100 µm . ( F ) Quantification of 1205Lu stable MCUA _ KD spheroid core size ( n = 6 biological replicates / condition , shown also by individual data points ) . Data information : Data are presented as mean ± SEM . Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test ( shMCU cells were compared to their respective control , shCTRL ) , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) . ( G ) Representative images of WM3734 shCTRL ( black frame ) , shMCU _ 1 ( darker orange frame ) and shMCU _ 2 ( lighter orange frame ) melanoma spheroids . Live cells are shown in green . Scale bar : 100 µm . ( H ) Quantification of WM3734 stable MCUA _ KD spheroid core size ( n = 5 biological replicates / condition , shown also by individual data points ) . Data information : Data are presented as mean ± SEM . Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test ( shMCU cells were compared to their respective control , shCTRL ) , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) . ( L ) Representative images of 451Lu wild - type ( grey frame ) , 451Lu - BR3 ( lighter green frame ) , WM983B wild - type ( grey frame ) and WM983B - BR ( darker green frame ) melanoma spheroids after 72 h invasion in collagen . Live cells are shown in green . Scale bar : 100 µm . ( M - N ) Quantification of 451Lu versus 451Lu - BR3 ( M ) and WM983B versus WM983B - BR ( N ) spheroid core size ( n ≥ 9 biological replicates / condition , shown also by individual data points ) . Data information : Data are presented as mean ± SEM . Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test ( shMCU cells were compared to their respective control , shCTRL ) , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) .

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Figure 3(C) Representative images of the migrated stained 1205Lu shCTRL (black frame), shMCU_1 (darker blue frame) and shMCU_2 (lighter blue frame) cells on the lower side of the insert. (D) Quantification of the transwell migration in 1205Lu stable MCUA_KD lines, based on the number of stained cells (n=4 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).(E) Representative images of 1205Lu shCTRL (black frame), shMCU_1 (darker blue frame) and shMCU_2 (lighter blue frame) melanoma spheroids after 72 h invasion in collagen. Live cells are shown in green. Scale bar: 100 µm. (F) Quantification of 1205Lu stable MCUA_KD spheroid core size (n=6 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).(G) Representative images of WM3734 shCTRL (black frame), shMCU_1 (darker orange frame) and shMCU_2 (lighter orange frame) melanoma spheroids. Live cells are shown in green. Scale bar: 100 µm. (H) Quantification of WM3734 stable MCUA_KD spheroid core size (n=5 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).(L) Representative images of 451Lu wild-type (grey frame), 451Lu-BR3 (lighter green frame), WM983B wild-type (grey frame) and WM983B-BR (darker green frame) melanoma spheroids after 72 h invasion in collagen. Live cells are shown in green. Scale bar: 100 µm. (M-N) Quantification of 451Lu versus 451Lu-BR3 (M) and WM983B versus WM983B-BR (N) spheroid core size (n≥9 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).