Representative H & E stainings of pancreata from KrasG12D and Hnf1apKO ; KrasG12D mice . B - D . KrasG12D and Hnf1apKO ; KrasG12D mice have normal morphology at 7 days . E - J . At 21 days Hnf1apKO ; KrasG12D mice show acinar - to - ductal metaplasia ( dashed encircled areas ) and regions with desmoplastic reaction ( asterisk ) , which are not observed in KrasG12D mice ( E , H ) . K - P . At 8 weeks KrasG12D pancreas show occasional abnormal ductal structures ( dashed encircled areas in N which is a magnification of squared dotted box in K ) and Hnf1apKO ; KrasG12D mice ( L , M , O , P ) present mucinous tubular complexes ( black arrows ) , and more advanced PanINs with luminal budding ( open arrows ) including foci of spindle cell proliferation ( asterisks ) and incipient infiltrative growth ( black dashed box area in O ) . Data information : Black dashed boxes in ( E , F , K , L and O ) indicate magnified areas in ( H , G , N , M and P ) respectively . Scale bars indicate 100 µm ( C , E , F , K , L ) , 50 µm ( O ) and 20 µm ( B , D , G , H - J , M , N , P ) .

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Representative H&E stainings of pancreata from KrasG12D and Hnf1apKO;KrasG12D mice. B-D. KrasG12D and Hnf1apKO;KrasG12D mice have normal morphology at 7 days. E-J. At 21 days Hnf1apKO;KrasG12D mice show acinar-to-ductal metaplasia (dashed encircled areas) and regions with desmoplastic reaction (asterisk), which are not observed in KrasG12D mice (E, H). K-P. At 8 weeks KrasG12D pancreas show occasional abnormal ductal structures (dashed encircled areas in N which is a magnification of squared dotted box in K) and Hnf1apKO;KrasG12D mice (L,M,O,P) present mucinous tubular complexes (black arrows), and more advanced PanINs with luminal budding (open arrows) including foci of spindle cell proliferation (asterisks) and incipient infiltrative growth (black dashed box area in O). Data information: Black dashed boxes in (E,F, K, L and O) indicate magnified areas in (H, G, N, M and P) respectively. Scale bars indicate 100 µm (C,E,F,K,L), 50 µm (O) and 20 µm (B,D,G,H-J,M,N,P).


A . Fold change ( FC ) in transcripts in Hnf1aaKO vs . control pancreas , plotted against significance ( - Log10 q ; genes significant at q < 0 . 05 are shown as colored dots above the horizontal line ) .

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A. Fold change (FC) in transcripts in Hnf1aaKO vs. control pancreas, plotted against significance (-Log10 q; genes significant at q<0.05 are shown as colored dots above the horizontal line).


B . GSEA showing that genes specific to differentiated acinar cells were downregulated in Hnf1aaKO pancreas , but not genes specific to islets or duct cells . Upregulated genes were enriched in genes specific to mesenchymal cells . Lineage - enriched genes were obtained from Muraro et al . ( Muraro et al . , 2016 ) .

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B. GSEA showing that genes specific to differentiated acinar cells were downregulated in Hnf1aaKO pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage-enriched genes were obtained from Muraro et al. (Muraro et al., 2016).


D . GSEA revealed that Hnf1aaKO pancreas showed increased transcripts involved in oncogenic pathways such as EMT , MAPK , KRAS , PI3K - AKT .

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D. GSEA revealed that Hnf1aaKO pancreas showed increased transcripts involved in oncogenic pathways such as EMT, MAPK, KRAS, PI3K-AKT.


E . HNF1A promotes transcriptional activation of direct target genes . Left : HNF1A bound - genes were enriched among genes that showed downregulation in Hnf1a mutants , but not among upregulated genes . P - values and odds ratios ( O . R . ) calculated by Fisher ' s exact test . Right : Venn diagrams showing overlap of HNF1A - bound genes with genes that were downregulated and upregulated in Hnf1a mutant pancreas .

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E. HNF1A promotes transcriptional activation of direct target genes. Left: HNF1A bound-genes were enriched among genes that showed downregulation in Hnf1a mutants, but not among upregulated genes. P-values and odds ratios (O.R.) calculated by Fisher's exact test. Right: Venn diagrams showing overlap of HNF1A-bound genes with genes that were downregulated and upregulated in Hnf1a mutant pancreas.


A . Human orthologs of genes that were up - and down - regulated in Hnf1aaKO pancreas were also up - and down - regulated in human pancreas with low vs . high HNF1A expression ( lowest vs . highest expression deciles , respectively ) . A random list of 717 genes controlled for similar expression levels was used for comparison . Violin plots include median and interquartile ranges . Dots are average values for each gene . Kruskall Wallis P < 0 . 0001 .

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A. Human orthologs of genes that were up- and down-regulated in Hnf1aaKO pancreas were also up- and down-regulated in human pancreas with low vs. high HNF1A expression (lowest vs. highest expression deciles, respectively). A random list of 717 genes controlled for similar expression levels was used for comparison. Violin plots include median and interquartile ranges. Dots are average values for each gene. Kruskall Wallis P<0.0001.


D . HNF1A mRNA levels differed in HNF1A LoF and control groups ( Kruskal - Wallis , P < 0 . 01 ) , despite considerable variability and overlap between groups . E . KDM6A mRNA levels were downregulated in HNF1A LoF tumors ( Kruskal - Wallis , P < 0 . 001 ) . Data information : Box plots in ( D and E ) show HNF1A and KDM6A expression in HNF1A LoF tumors ( n = 26 ) and Control 1 ( n = 39 ) and Control 2 ( n = 57 ) tumors . The horizontal central line marks the median . Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1 . 5 x interquartile range outside box limits .

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D. HNF1A mRNA levels differed in HNF1A LoF and control groups (Kruskal-Wallis, P<0.01), despite considerable variability and overlap between groups. E. KDM6A mRNA levels were downregulated in HNF1A LoF tumors (Kruskal-Wallis, P<0.001).Data information: Box plots in (D and E) show HNF1A and KDM6A expression in HNF1A LoF tumors (n=26) and Control 1 (n=39) and Control 2 (n=57) tumors. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x interquartile range outside box limits.


H & E staining of pancreata from Kdm6apKO ; KrasG12D and KrasG12D mice . A - C . 7 day - old KrasG12D and Kdm6apKO ; KrasG12D mice showed normal morphology . D - I . At 21 days , KrasG12D mice showed normal pancreas morphology ( dotted area at in D is shown at large magnification in G ) whereas Kdm6apKO ; KrasG12D mice already show acinar - to - ductal metaplasia ( dashed encircled area in F ) , spindle cell proliferation ( asterisks in E and H ) , sarcomatoid architecture ( I ) and desmoplastic reaction ( black arrowhead in H ) . J - O . KrasG12D mice present occasional acinar - to - ductal metaplasia and low grade PanINs at 8 weeks ( M is a magnification of squared dotted box in J and see Fig 1N ) whereas at the same age pancreas from Kdm6apKO ; KrasG12D mice show massive remodeling ( K ) , extensive acinar - to - ductal metaplasia ( L and dashed encircled area in N ) , cancer with prominent spindle / mesenchymal proliferation , infiltrative growth ( black arrows and asterisk respectively in O ) and abundant stroma ( black arrowheads in N ) . Data information : Scale bars : 100 µm ( B , D , E , J , K ) or 20 µm ( A , C , F - I , L - N ) .

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H&E staining of pancreata from Kdm6apKO;KrasG12D and KrasG12D mice. A-C. 7 day-old KrasG12D and Kdm6apKO;KrasG12D mice showed normal morphology. D-I. At 21 days, KrasG12D mice showed normal pancreas morphology (dotted area at in D is shown at large magnification in G) whereas Kdm6apKO;KrasG12D mice already show acinar-to-ductal metaplasia (dashed encircled area in F), spindle cell proliferation (asterisks in E and H), sarcomatoid architecture (I) and desmoplastic reaction (black arrowhead in H). J-O. KrasG12D mice present occasional acinar-to-ductal metaplasia and low grade PanINs at 8 weeks (M is a magnification of squared dotted box in J and see Fig 1N) whereas at the same age pancreas from Kdm6apKO;KrasG12D mice show massive remodeling (K), extensive acinar-to-ductal metaplasia (L and dashed encircled area in N), cancer with prominent spindle/mesenchymal proliferation, infiltrative growth (black arrows and asterisk respectively in O) and abundant stroma (black arrowheads in N). Data information: Scale bars: 100 µm (B,D,E,J,K) or 20 µm (A,C,F-I, L-N).


A . Fold change ( FC ) and significance for transcripts in Kdm6apKO vs . control pancreas . Genes significant at q < 0 . 05 are shown as colored dots above the horizontal line .

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A. Fold change (FC) and significance for transcripts in Kdm6apKO vs. control pancreas. Genes significant at q<0.05 are shown as colored dots above the horizontal line.


B . GSEA showing that genes specific to differentiated acinar cells were downregulated in KDM6A - deficient pancreas , but not genes specific to islets or duct cells . Upregulated genes were enriched in genes specific to mesenchymal cells . Lineage - enriched genes were obtained from ( Muraro et al . , 2016 ) .

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B. GSEA showing that genes specific to differentiated acinar cells were downregulated in KDM6A-deficient pancreas, but not genes specific to islets or duct cells. Upregulated genes were enriched in genes specific to mesenchymal cells. Lineage-enriched genes were obtained from (Muraro et al., 2016).


D . GSEA showed increased expression of indicated oncogenic pathway genes in Kdm6apKO pancreas .

D. GSEA showed increased expression of indicated oncogenic pathway genes in Kdm6apKO pancreas.


E . KDM6A functions as a transcriptional activator of direct target genes in pancreatic cells . Left : Downregulated , but not upregulated , genes in Kdm6apKO pancreas were enriched for KDM6A binding . P - values and odds ratios ( O . R . ) were calculated by Fisher ' s exact test . Right : Venn diagrams showing overlap of down and upregulated genes with KDM6A binding .

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E. KDM6A functions as a transcriptional activator of direct target genes in pancreatic cells. Left: Downregulated, but not upregulated, genes in Kdm6apKO pancreas were enriched for KDM6A binding. P-values and odds ratios (O.R.) were calculated by Fisher's exact test. Right: Venn diagrams showing overlap of down and upregulated genes with KDM6A binding.


F . KDM6A - bound enhancers and promoters showed increased H3K27me3 and decreased H3K27ac in Kdm6apKO pancreas .

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F. KDM6A-bound enhancers and promoters showed increased H3K27me3 and decreased H3K27ac in Kdm6apKO pancreas.


G . Genes that were downregulated in Kdm6apKO pancreas ( KDM6A - dependent genes ) showed greatest changes in histone marks . Box plots show fold - changes of H3K27me3 ( left ) and H3K27ac ( right ) signals in Kdm6apKO compared to control . Signals were analyzed in KDM6A bound regions in promoters and enhancers of genes that were KDM6A dependent ( grey ; n = 420 regions ) and independent ( white ; n = 8035 regions ) . The signals are average values from ChIP - seq experiments in two biological replicates . The horizontal central line marks the median . Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1 . 5 x IQR outside box limits . P - values were determined by two - tailed Mann - Whitney U test .

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G. Genes that were downregulated in Kdm6apKO pancreas (KDM6A-dependent genes) showed greatest changes in histone marks. Box plots show fold-changes of H3K27me3 (left) and H3K27ac (right) signals in Kdm6apKO compared to control. Signals were analyzed in KDM6A bound regions in promoters and enhancers of genes that were KDM6A dependent (grey; n=420 regions) and independent (white; n=8035 regions). The signals are average values from ChIP-seq experiments in two biological replicates. The horizontal central line marks the median. Box limits indicate the first and third quartiles and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Mann-Whitney U test.


H . Changes in KDM6A , H3K27me3 and H3K27ac ChIP - seq profiles in the Ppp4r4 - Serpina10 locus in Kdm6apKO pancreas .

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H. Changes in KDM6A, H3K27me3 and H3K27ac ChIP-seq profiles in the Ppp4r4-Serpina10 locus in Kdm6apKO pancreas.


I . qPCR of H3K27me3 , H3K27ac and H3K4me1 changes in promoter and enhancer regions highlighted in ( H ) ( R1 , R2 , R3 ) , and Serpin10 mRNA . Error bars show + / - SD and P - values are from two - tailed Student ' s t test , n = 3 .

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I. qPCR of H3K27me3, H3K27ac and H3K4me1 changes in promoter and enhancer regions highlighted in (H) (R1, R2, R3), and Serpin10 mRNA. Error bars show +/-SD and P-values are from two-tailed Student's t test, n=3.


J . KDM6A - bound genes that showed decreased expression in Kdm6apKO pancreas ( functional KDM6A binding sites ) showed simultaneous gain of H3K27me3 and loss and of H3K27ac .

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J. KDM6A-bound genes that showed decreased expression in Kdm6apKO pancreas (functional KDM6A binding sites) showed simultaneous gain of H3K27me3 and loss and of H3K27ac.


B . Co - binding analysis in functional KDM6A - bound enhancer and promoter regions revealed that HNF1A was the most enriched co - bound TF amongst three other acinar cell TFs . Binding regions of TAL1 in a non - pancreatic cell type and random binding sites were used as negative controls . P - values was determined by Fisher ' s exact test for peak comparisons using all enhancer and promoter regions as background .

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B. Co-binding analysis in functional KDM6A-bound enhancer and promoter regions revealed that HNF1A was the most enriched co-bound TF amongst three other acinar cell TFs. Binding regions of TAL1 in a non-pancreatic cell type and random binding sites were used as negative controls. P-values was determined by Fisher's exact test for peak comparisons using all enhancer and promoter regions as background.


C . The most downregulated genes in Kdm6apKO pancreas are shown ranked by q - value , and are almost invariably bound by HNF1A and down regulated in Hnf1aaKO pancreas , or known to be direct HNF1A - dependent target genes from other studies ( red and purple , respectively ) .

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C. The most downregulated genes in Kdm6apKO pancreas are shown ranked by q-value, and are almost invariably bound by HNF1A and down regulated in Hnf1aaKO pancreas, or known to be direct HNF1A-dependent target genes from other studies (red and purple, respectively).


D , E . GSEA analysis on the Hnf1aaKO and Kdm6apKO ranked - ordered gene lists vs . their reciprocal up - or downregulated gene sets , demonstrated that KDM6A and HNF1A regulate similar genes .

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D,E. GSEA analysis on the Hnf1aaKO and Kdm6apKO ranked-ordered gene lists vs. their reciprocal up- or downregulated gene sets, demonstrated that KDM6A and HNF1A regulate similar genes.


F . Expression changes in Hnf1aaKO and Kdm6apKO pancreas , showing that genes bound by KDM6A and downregulated in Kdm6apKO pancreas ( red dots ) were generally downregulated in Hnf1aaKO pancreas .

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F. Expression changes in Hnf1aaKO and Kdm6apKO pancreas, showing that genes bound by KDM6A and downregulated in Kdm6apKO pancreas (red dots) were generally downregulated in Hnf1aaKO pancreas.


G . HNF1A and KDM6A co - occupy the same regions in Pah , which is downregulated in Hnf1a and Kdm6a knock - out pancreas .

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G. HNF1A and KDM6A co-occupy the same regions in Pah, which is downregulated in Hnf1a and Kdm6a knock-out pancreas.


H . Genes that were co - bound by KDM6A and HNF1A showed greatest downregulation in Kdm6apKO pancreas , compared with KDM6A - bound genes that were not bound by HNF1A . Box plots show median and IQR of Log2 TPM fold - changes and whiskers extend to highest and lowest data points within 1 . 5 x IQR outside box limits . P - values were determined by two - tailed Student ' s t tests and n = 4 replicates per condition .

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H. Genes that were co-bound by KDM6A and HNF1A showed greatest downregulation in Kdm6apKO pancreas, compared with KDM6A-bound genes that were not bound by HNF1A. Box plots show median and IQR of Log2 TPM fold-changes and whiskers extend to highest and lowest data points within 1.5 x IQR outside box limits. P-values were determined by two-tailed Student's t tests and n=4 replicates per condition.


A . Co - immunoprecipitation of endogenous HNF1A and KDM6A followed by western blot demonstrated that HNF1A is in the same complex as KDM6A .

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A. Co-immunoprecipitation of endogenous HNF1A and KDM6A followed by western blot demonstrated that HNF1A is in the same complex as KDM6A.


B . Western blot showing loss of HNF1A and unchanged KDM6A in Hnf1a - / - pancreas .

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B. Western blot showing loss of HNF1A and unchanged KDM6A in Hnf1a-/- pancreas.


C . Differential binding analysis of KDM6A in Hnf1a - / - vs . wild type pancreas . Pink dots below zero ( 1873 sites ) show regions with reduced KDM6A binding and pink dots above zero ( 118 sites ) are regions with increased binding at FDR < 0 . 05 .

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C. Differential binding analysis of KDM6A in Hnf1a-/- vs. wild type pancreas. Pink dots below zero (1873 sites) show regions with reduced KDM6A binding and pink dots above zero (118 sites) are regions with increased binding at FDR<0.05.


Regions that show reduced KDM6A binding in Hnf1a - / - chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs . P - values in ( D ) were calculated with two - tailed Mann - Whitney U

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Regions that show reduced KDM6A binding in Hnf1a-/- chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs. P-values in (D) were calculated with two-tailed Mann-Whitney U test


Regions that show reduced KDM6A binding in Hnf1a - / - chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs . P - values in ( E ) with Fisher ' s exact test .

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Regions that show reduced KDM6A binding in Hnf1a-/- chromatin are strongly bound by HNF1A and are highly enriched in HNF1 motifs. P-values in (E) with Fisher's exact test.


F . KDM6A binding is markedly reduced in HNF1A and KDM6A co - bound regions in Hnf1a - / - pancreas , but not in other KDM6A bound regions .

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F. KDM6A binding is markedly reduced in HNF1A and KDM6A co-bound regions in Hnf1a-/- pancreas, but not in other KDM6A bound regions.


G . Genes that loose KDM6A binding in Hnf1a - mutant pancreas are predominantly downregulated in Hnf1aaKO pancreas and are direct HNF1A target genes ( red dots ) .

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G. Genes that loose KDM6A binding in Hnf1a-mutant pancreas are predominantly downregulated in Hnf1aaKO pancreas and are direct HNF1A target genes (red dots).


The violin plot depicting the smaller genome size of HAC isolates ( n = 820 ) than that of DPC ( n = 407 ) and HPC ( n = 262 ) . Inside the violins are the box and whisker plots , with the dot showing the median , the box showing the quartiles , and the whisker showing the 95 % percentiles . P - values were calculated using unpaired t - test ( * * , P < 0 . 05 ) .

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The violin plot depicting the smaller genome size of HAC isolates (n=820) than that of DPC (n=407) and HPC (n=262). Inside the violins are the box and whisker plots, with the dot showing the median, the box showing the quartiles, and the whisker showing the 95% percentiles. P-values were calculated using unpaired t-test (**, P<0.05).


The survival curve for zebrafish inoculated with representative S . suis isolates from HAC and HPC . In total twenty - five representative isolates were tested , including fourteen isolates from HAC and ten healthy - pig isolates from HPC , as well as the strain SC19 in HAC responsible for the 2005 human outbreak as a positive control . The cumulative mortalities were calculated using the combined data from the isolates in each group . As expected , all 14 isolates from HAC displayed a significantly higher mortality than the HPC isolates ( P < 0 . 05 ) . Survival curve and statistical analysis were created using Kaplan - Meier and SPSS 23 . 0 . P - values were calculated using Log rank test . P < 0 . 05 was considered significant ( * * , P < 0 . 05 ; NS , P > 0 . 05 ) . No significant difference ( NS , P = 0 . 072 ) was found between HAC isolates and SC19 .

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The survival curve for zebrafish inoculated with representative S. suis isolates from HAC and HPC. In total twenty-five representative isolates were tested, including fourteen isolates from HAC and ten healthy-pig isolates from HPC, as well as the strain SC19 in HAC responsible for the 2005 human outbreak as a positive control. The cumulative mortalities were calculated using the combined data from the isolates in each group. As expected, all 14 isolates from HAC displayed a significantly higher mortality than the HPC isolates (P<0.05). Survival curve and statistical analysis were created using Kaplan-Meier and SPSS 23.0. P-values were calculated using Log rank test. P<0.05 was considered significant (**, P<0.05; NS, P>0.05). No significant difference (NS, P=0.072) was found between HAC isolates and SC19.


A ) Shear - dilation relations of Tek - Cre : : Lama5 - / - and Lama4 - / - mesenteric resistance arteries and corresponding wild type controls show a reduced response of Tek - Cre : : Lama5 - / - arteries and an enhanced response of Lama4 - / - arteries . Data shown are mean changes in vessel diameter ( Δ µm ) ± s . e . m . from 8 experiments with 16 wild type and 16 KO arteries ; KO and wild type were analysed as pairs in each experiment . * * P < 0 . 01 , paired t - test .

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A) Shear-dilation relations of Tek-Cre::Lama5-/- and Lama4-/- mesenteric resistance arteries and corresponding wild type controls show a reduced response of Tek-Cre::Lama5-/- arteries and an enhanced response of Lama4-/- arteries. Data shown are mean changes in vessel diameter (Δ µm) ± s.e.m. from 8 experiments with 16 wild type and 16 KO arteries; KO and wild type were analysed as pairs in each experiment. **P<0.01, paired t-test.


The dose - response curves of arteries from wild type , Tek - Cre : : Lama5 - / - ( B ) and Lama4 - / - mice ( C ) and wild type littermates stimulated with methacholine do not show significant differences ( n . s . ) . Data are expressed as percent relaxation of the maximum force developed in presence of 0 . 3 µM U46619 , and are mean values ± s . e . m from 7 experiments with 1 wild type and 1 KO artery in each experiment .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The dose-response curves of arteries from wild type, Tek-Cre::Lama5-/- (B) and Lama4-/- mice (C) and wild type littermates stimulated with methacholine do not show significant differences (n.s.). Data are expressed as percent relaxation of the maximum force developed in presence of 0.3 µM U46619, and are mean values ± s.e.m from 7 experiments with 1 wild type and 1 KO artery in each experiment.


A ) Three - dimensional digital reconstructions of optical sections through immunofluorescently stained mesenteric resistance arteries from wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice , and optical sections through vessel walls to show the endothelial and underlying smooth muscle layers . DAPI staining permits identification of endothelial cell nuclei , which lie perpendicular to the nuclei of the smooth muscle cells . Laminin α5 is absent from endothelial basement membranes of Tek - Cre : : Lama5 - / - arteries ( arrow ) but is still present in smooth muscle basement membranes ( asterisk ) , while laminin α4 is still detectable in both endothelial and smooth muscle basement membranes . Lama4 - / - arteries lack laminin α4 in both endothelial ( arrow ) and smooth muscle ( asterisk ) basement membranes , but laminin α5 is still detectable . Scale bars are 10 μm .

O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O

A) Three-dimensional digital reconstructions of optical sections through immunofluorescently stained mesenteric resistance arteries from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice, and optical sections through vessel walls to show the endothelial and underlying smooth muscle layers. DAPI staining permits identification of endothelial cell nuclei, which lie perpendicular to the nuclei of the smooth muscle cells. Laminin α5 is absent from endothelial basement membranes of Tek-Cre::Lama5-/- arteries (arrow) but is still present in smooth muscle basement membranes (asterisk), while laminin α4 is still detectable in both endothelial and smooth muscle basement membranes. Lama4-/- arteries lack laminin α4 in both endothelial (arrow) and smooth muscle (asterisk) basement membranes, but laminin α5 is still detectable. Scale bars are 10 μm.


B ) Scanning electron microscopy images of endothelial cell - denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - arteries . The arrows indicate artificial ruptures due to preparation procedure . Scale bars are 100 nm .

O O O O O O O O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

B) Scanning electron microscopy images of endothelial cell-denuded mesenteric resistance arteries show a comparable topography of the endothelial basement membrane in wild type, Tek-Cre::Lama5-/- and Lama4-/- arteries. The arrows indicate artificial ruptures due to preparation procedure. Scale bars are 100 nm.


C ) Intravital microscopic quantification of mesenteric arteries sizes in vivo show smaller diameters ( - 24 . 3 % ) in Tek - Cre : : Lama5 - / - arteries and larger diameters ( + 27 . 6 % ) in Lama4 - / - arteries compared to wild type controls . Auto - fluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter . Scale bar is 100 µm . Data are means ± s . e . m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype . * P < 0 . 05 unpaired t - test .

O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Intravital microscopic quantification of mesenteric arteries sizes in vivo show smaller diameters (-24.3%) in Tek-Cre::Lama5-/- arteries and larger diameters (+27.6%) in Lama4-/- arteries compared to wild type controls. Auto-fluorescence of the internal elastic lamina allowed a good approximation of mesenteric arteries lumen diameter. Scale bar is 100 µm. Data are means ± s.e.m from a minimum of 4 first order mesenteric arteries imaged at least in 4 mice per genotype. * P<0.05 unpaired t-test.


A ) In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511 , compared to the non - endothelial cell laminin 111 and fibronectin , showing high levels of HUAECs adhesion to laminin 511 and low binding to laminin 411 .

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A) In vitro cell adhesion assays employing HUAECs plated on increasing concentrations of purified laminin 411 and 511, compared to the non-endothelial cell laminin 111 and fibronectin, showing high levels of HUAECs adhesion to laminin 511 and low binding to laminin 411.


B ) Enhanced fold change ( Δ ) in COX2 mRNA expression in HUAECs plated on laminin 511 versus laminin 111 in response to shear . mRNA data are means ± s . e . m of 4 independent experiments with triplicates / experiment , paired t - test .

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B) Enhanced fold change (Δ) in COX2 mRNA expression in HUAECs plated on laminin 511 versus laminin 111 in response to shear. mRNA data are means ± s.e.m of 4 independent experiments with triplicates/ experiment, paired t-test.


B ) Enhanced fold change ( Δ ) in COX2 protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear . Protein quantification data are means ± s . e . m of 7 independent experiments , Mann - Whitney test .

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B) Enhanced fold change (Δ) in COX2 protein levels in HUAECs plated on laminin 511 versus laminin 111 in response to shear. Protein quantification data are means ± s.e.m of 7 independent experiments, Mann-Whitney test.


C ) Angle histogram of HUAEC orientation after 120 min of 10dyn / cm2 shear stress showing the percent cells at 10 - 90 ° in relation to the direction of flow . Cells were plated on 25 nM laminin 511 and laminin 111 . Data are means ± s . e . m of 6 independent experiments , t - test .

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C) Angle histogram of HUAEC orientation after 120 min of 10dyn/cm2 shear stress showing the percent cells at 10-90° in relation to the direction of flow. Cells were plated on 25 nM laminin 511 and laminin 111. Data are means ± s.e.m of 6 independent experiments, t-test.


D , E ) Inhibition assays performed at 25nM laminin 511 ( D ) in the presence or absence of function blocking integrin antibodies . Data are means ± s . e . m of 4 experiments with triplicates / experiment , unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

D, E) Inhibition assays performed at 25nM laminin 511 (D) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.


D , E ) Inhibition assays performed at 25nM laminin 111 , laminin 411 or fibronectin ( E ) in the presence or absence of function blocking integrin antibodies . Data are means ± s . e . m of 4 experiments with triplicates / experiment , unpaired t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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D, E) Inhibition assays performed at 25nM laminin 111, laminin 411 or fibronectin (E) in the presence or absence of function blocking integrin antibodies. Data are means ± s.e.m of 4 experiments with triplicates/ experiment, unpaired t-test . *P<0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.


( A ) Double immunofluorescence staining of whole mount wild type mesenteric resistance arteries for β1 integrin and vinculin show colocalization in focal adhesions ( arrow heads ) . Scale bar is 10 µm .

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(A) Double immunofluorescence staining of whole mount wild type mesenteric resistance arteries for β1 integrin and vinculin show colocalization in focal adhesions (arrow heads). Scale bar is 10 µm.


B ) Vinculin immunofluorescence staining of whole mount wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek - Cre : : Lama5 - / - ( arrow heads ) and larger adhesion complexes in Lama4 - / - vessels ( arrow heads ) . Scale bar is 10 µm . Data are means ± s . e . m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice / genotype . * * * P < 0 . 001 , * * * * P < 0 . 0001 , unpaired t - test .

O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Vinculin immunofluorescence staining of whole mount wild type, Tek-Cre::Lama5-/- and Lama4-/- mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek-Cre::Lama5-/- (arrow heads) and larger adhesion complexes in Lama4-/- vessels (arrow heads). Scale bar is 10 µm. Data are means ± s.e.m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice/genotype. ***P<0.001, ****P<0.0001, unpaired t-test.


B ) Vinculin immunofluorescence staining of whole mount wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek - Cre : : Lama5 - / - ( arrow heads ) and larger adhesion complexes in Lama4 - / - vessels ( arrow heads ) . Scale bar is 10 µm . Correpsonding quantification of sizes ( C ) and frequency distribution ( D ) of adhesion complexes per endothelial cell . Data are means ± s . e . m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice / genotype . * * * P < 0 . 001 , * * * * P < 0 . 0001 , unpaired t - test .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Vinculin immunofluorescence staining of whole mount wild type, Tek-Cre::Lama5-/- and Lama4-/- mice mesenteric resistance arteries reveal smaller adhesion complexes in Tek-Cre::Lama5-/- (arrow heads) and larger adhesion complexes in Lama4-/- vessels (arrow heads). Scale bar is 10 µm. Correpsonding quantification of sizes (C) and frequency distribution (D) of adhesion complexes per endothelial cell. Data are means ± s.e.m from 300 cells from 9 wild type and 9 KO arteries isolated from 3 mice/genotype. ***P<0.001, ****P<0.0001, unpaired t-test.


E , F ) The endothelium of excised wild type , Tek - Cre : : Lama5 - / - aortae were analysed by AFM , revealing reduced cortical stiffness in Tek - Cre : : Lama5 - / - vessels ( - 9 . 5 % ) . Data are means ± s . e . m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment . * P < 0 . 05 , * * * * P < 0 . 0001 , unpaired t - test .

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E, F) The endothelium of excised wild type, Tek-Cre::Lama5-/- aortae were analysed by AFM, revealing reduced cortical stiffness in Tek-Cre::Lama5-/- vessels (-9.5%). Data are means ± s.e.m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment. *P<0.05, ****P<0.0001, unpaired t-test.


E , F ) The endothelium of excised wild type and Lama4 - / - aortae were analysed by AFM , revealing increased cortical stiffness in Lama4 - / - vessels ( + 2 % ) . Data are means ± s . e . m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment . * P < 0 . 05 , * * * * P < 0 . 0001 , unpaired t - test .

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E, F) The endothelium of excised wild type and Lama4-/- aortae were analysed by AFM, revealing increased cortical stiffness in Lama4-/- vessels (+2%). Data are means ± s.e.m from 4 experiments employing 4 KO arteries and 4 wild type controls in each experiment. *P<0.05, ****P<0.0001, unpaired t-test.


G ) In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111 , reveal increased cortical stiffness in cells on laminin 511 . Data are mean ± s . e . m from 3 experiments with triplicates / experiments , * P < 0 . 05 , unpaired t - test .

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G) In vitro AFM measurements of cortical stiffness performed on HUAECs plated on 30 nM purified laminin 511 or 111, reveal increased cortical stiffness in cells on laminin 511. Data are mean ± s.e.m from 3 experiments with triplicates/experiments, *P<0.05, unpaired t-test.


A ) A dual pipette - pulling assay was employed to measure cell - cell adhesion strength in HUAECs bound to laminin - coated beads ( asterisk ) or in the absence of beads . Scale bar is 10 µm . B ) Quantification shows higher adhesion strength between cells bound to laminin 511 - coated beads compared to laminin 111 - coated beads or cells not incubated with beads . Measurements could not be made with cells incubated with laminin 411 - coated beads since it was impossible to find cell - bead complexes . Values are means ± s . e . m from 15 cells from 3 independent experiments , unpaired t - test with Welch ' s correction .

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A) A dual pipette-pulling assay was employed to measure cell-cell adhesion strength in HUAECs bound to laminin-coated beads (asterisk) or in the absence of beads. Scale bar is 10 µm. B) Quantification shows higher adhesion strength between cells bound to laminin 511-coated beads compared to laminin 111-coated beads or cells not incubated with beads. Measurements could not be made with cells incubated with laminin 411-coated beads since it was impossible to find cell-bead complexes. Values are means ± s.e.m from 15 cells from 3 independent experiments, unpaired t-test with Welch's correction.


C ) Dual pipette - pulling assay performed in the presence of VE - cadherin blocking antibody , isotope control and soluble laminin 511 , showing almost complete ablation of adhesion strength in the presence of the blocking antibody in all conditions . Similar adhesion strengths were measured between cells in the presence of soluble laminin 511 and cells bound to laminin 511 - coated beads .

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C) Dual pipette-pulling assay performed in the presence of VE-cadherin blocking antibody, isotope control and soluble laminin 511, showing almost complete ablation of adhesion strength in the presence of the blocking antibody in all conditions. Similar adhesion strengths were measured between cells in the presence of soluble laminin 511 and cells bound to laminin 511-coated beads.


D ) Adhesion strength in the presence of soluble laminin 511 was significantly reduced in the presence of integrin β1 blocking antibody ; cell - cell adhesion strength between cells bound to laminin 511 coated beads was significantly reduced in the presence of ROCK , SRC or FAK inhibitors . Values are means ± s . e . m from 11 cells from 3 independent experiments , unpaired t - test with Welch ' s correction . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , n . s . = not significant , n . d . = not determined .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D) Adhesion strength in the presence of soluble laminin 511 was significantly reduced in the presence of integrin β1 blocking antibody; cell-cell adhesion strength between cells bound to laminin 511 coated beads was significantly reduced in the presence of ROCK, SRC or FAK inhibitors. Values are means ± s.e.m from 11 cells from 3 independent experiments, unpaired t-test with Welch's correction. *P <0.05, **P<0.01, ***P<0.001, ****P<0.0001, n.s. = not significant, n.d. = not determined.


A ) Immunofluorescence - based antibody feeding assay performed using HUAECs seeded on laminin 411 , 511 or 111 - coated coverslips , reveals more VE - cadherin at junctions and less in vesicles in cells plated on laminin 511 ( arrow heads mark VE - cadherin positive vesicles ) . Scale bar is 10 µm . B ) Quantification of VE - cadherin positive vesicles / cell in HUAECs plated on laminin 411 , 511 or 111 . Values are means ± s . e . m from 300 cells from 3 independent experiments , unpaired t - test .

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A) Immunofluorescence-based antibody feeding assay performed using HUAECs seeded on laminin 411, 511 or 111-coated coverslips, reveals more VE-cadherin at junctions and less in vesicles in cells plated on laminin 511 (arrow heads mark VE-cadherin positive vesicles). Scale bar is 10 µm. B) Quantification of VE-cadherin positive vesicles/ cell in HUAECs plated on laminin 411, 511 or 111. Values are means ± s.e.m from 300 cells from 3 independent experiments, unpaired t-test.


C ) Western blot of biotinylated ( surface ) VE - cadherin in HUAECs plated on different laminins versus total VE - cadherin ( input ) confirms more junctionally located VE - cadherin in cells bound to laminin 511 .

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C) Western blot of biotinylated (surface) VE-cadherin in HUAECs plated on different laminins versus total VE-cadherin (input) confirms more junctionally located VE-cadherin in cells bound to laminin 511.


D ) Quantification of Western blots expressed as relative signal proportions ( surface VE - cadherin / total VE - cadherin ) . Values shown are means ± s . e . m of 6 experiments , unpaired t - test .

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D) Quantification of Western blots expressed as relative signal proportions (surface VE-cadherin/ total VE-cadherin). Values shown are means ± s.e.m of 6 experiments, unpaired t-test.


E ) Western blot of p120 catenincoimmunoprecipitated with VE - cadherin ( input ) from HUAECs plated on different laminins Asterisks are isotope controls . Values shown are means ± s . e . m of 4 experiments , Mann - Whitney test .

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E) Western blot of p120 catenincoimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.


E ) Western blot of p120 catenin coimmunoprecipitated with VE - cadherin ( input ) from HUAECs plated on different laminins , and ( F ) corresponding quantification of the p120 signal intensity relative to the total VE - cadherin signal . Asterisks are isotope controls . Values shown are means ± s . e . m of 4 experiments , Mann - Whitney test .

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E) Western blot of p120 catenin coimmunoprecipitated with VE-cadherin (input) from HUAECs plated on different laminins, and (F) corresponding quantification of the p120 signal intensity relative to the total VE-cadherin signal. Asterisks are isotope controls. Values shown are means ± s.e.m of 4 experiments, Mann-Whitney test.


A ) Double immunofluorescence staining for phosphorylated myosin light chain II ( pMLC II ) and VE - cadherin in mesenteric resistance artery endothelium from wild type , Tek - Cre : : Lama5 - / - and Lama4 - / - mice ( arrow heads ) . Scale bar is 10µm . B ) Quantification of the pMLC II signal along VE - cadherin signal revealed significantly less overlap in Tek - Cre : : Lama5 - / - and more overlap in Lama4 - / - endothelium , compared with wild type controls . The data are normalized to the skeletal length of the VE - cadherin signal . Values shown are means ± s . e . m resulting from quantification of 4 field of view per arteries , 4 mice / genotype . * P < 0 . 05 , * * P < 0 . 01 , paired t - test .

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A) Double immunofluorescence staining for phosphorylated myosin light chain II (pMLC II) and VE-cadherin in mesenteric resistance artery endothelium from wild type, Tek-Cre::Lama5-/- and Lama4-/- mice (arrow heads). Scale bar is 10µm. B) Quantification of the pMLC II signal along VE-cadherin signal revealed significantly less overlap in Tek-Cre::Lama5-/- and more overlap in Lama4-/- endothelium, compared with wild type controls. The data are normalized to the skeletal length of the VE-cadherin signal. Values shown are means ± s.e.m resulting from quantification of 4 field of view per arteries, 4 mice /genotype. *P<0.05, **P<0.01, paired t-test.


A , B . Aggregate number of detected and undetected cysteines ( A ) and lysines ( B ) in 3 , 840 CpDAA - containing proteins .

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A, B. Aggregate number of detected and undetected cysteines (A) and lysines (B) in 3,840 CpDAA-containing proteins.


C . Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220 - 479 of CASP8 ( UniProt ID Q14790 ) . Dashed horizontal line marks deleterious threshold of 25 .

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C. Shows CADD38 max codon scores for nonsynonymous SNVs at residues 220-479 of CASP8 (UniProt ID Q14790). Dashed horizontal line marks deleterious threshold of 25.


E . Activity of recombinant caspase - 8 protein assayed using fluorogenic IETD - AFC substrate . Percentage activity shown relative to wild - type ( WT ) protein for three replicate experiments , bars and error bars as mean ± SD .

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E. Activity of recombinant caspase-8 protein assayed using fluorogenic IETD-AFC substrate. Percentage activity shown relative to wild-type (WT) protein for three replicate experiments, bars and error bars as mean ± SD.


( A ) Amylose pulldown from benzonase - treated HEK293T cell lysates expressing 2xMBP - BRCA2NT in untreated or irradiated cells ( 6Gy ; + IR ) . DDX5 and BRCA2NT ( MBP ) detected by immunoblot . StainFree images of the gels before transfer were used as loading

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(A) Amylose pulldown from benzonase-treated HEK293T cell lysates expressing 2xMBP-BRCA2NT in untreated or irradiated cells (6Gy; +IR). DDX5 and BRCA2NT (MBP) detected by immunoblot. StainFree images of the gels before transfer were used as loading control


( B ) Immunoprecipitation ( IP ) of endogenous BRCA2 from benzonase - treated HEK293T cell lysates left untreated or treated with IR ( 6Gy ) and harvested 4h post - IR , as indicated . Mouse IgG was used as negative control . Immunoblot of DDX5 and BRCA2 . StainFree image of the gels before transfer were used as loading control ( cropped image is shown ) . Asterisk ( * ) indicate a non - specific band detected by anti - DDX5 antibody .

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(B) Immunoprecipitation (IP) of endogenous BRCA2 from benzonase-treated HEK293T cell lysates left untreated or treated with IR (6Gy) and harvested 4h post-IR, as indicated. Mouse IgG was used as negative control. Immunoblot of DDX5 and BRCA2. StainFree image of the gels before transfer were used as loading control (cropped image is shown). Asterisk (*) indicate a non-specific band detected by anti-DDX5 antibody.


( C ) Left : Representative images of in situ proximity ligation assay ( PLA ) between BRCA2 and DDX5 antibodies in U2OS cells either left untreated ( - ) or irradiated ( 4h post - IR ; 6 Gy ) . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . When indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) 24h before or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls from untreated siC cells are shown . Scale bar indicates 10 µm . Right : Quantification of the number of PLA spots per nucleus . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

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(C) Left: Representative images of in situ proximity ligation assay (PLA) between BRCA2 and DDX5 antibodies in U2OS cells either left untreated (-) or irradiated (4h post-IR; 6 Gy). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from untreated siC cells are shown. Scale bar indicates 10 µm. Right: Quantification of the number of PLA spots per nucleus. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( D ) Diagram showing the BRCA2 N - terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N - terminal truncations ( BRCA2T1 , BRCA2LT2 , BRCA2LT3 ) or the 2xMBP tag . DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP , respectively . StainFree images of the gels before transfer were used as loading

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(D) Diagram showing the BRCA2 N-terminal truncations used in this study and amylose pulldown from HEK293T whole cells extracts overexpressing the indicated BRCA2 N-terminal truncations (BRCA2T1, BRCA2LT2, BRCA2LT3) or the 2xMBP tag. DDX5 and BRCA2 truncations were detected using specific antibodies against DDX5 and MBP, respectively. StainFree images of the gels before transfer were used as loading control


( E ) Left : GST pulldown assay using purified BRCA2T1 and DDX5 ; MBP antibody was used for the detection of both proteins . UB : unbound ; E : eluate . Right : SDS - PAGE showing 300 ng of purified MBP - DDX5 - GST and of 2xMBP - tagged BRCA2T1 used in the pulldown assay .

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(E) Left: GST pulldown assay using purified BRCA2T1 and DDX5; MBP antibody was used for the detection of both proteins. UB: unbound; E: eluate. Right: SDS-PAGE showing 300 ng of purified MBP-DDX5-GST and of 2xMBP-tagged BRCA2T1 used in the pulldown assay.


( A ) Left : Representative images of S9 . 6 immunofluorescence of U2OS cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) after transfection with either an empty plasmid or a plasmid expressing DDX5 . The merged images show the signal of S9 . 6 , nucleolin ( nucleoli ) antibodies and DAPI staining . Scale bar indicates 25 µm . Right : Quantification of S9 . 6 average nuclear intensity of U2OS cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) after transfection with either an empty plasmid or a plasmid expressing DDX5 . The red line in the plot indicates the median and each symbol represents the value of a single cell . The statistical significance of the difference was calculated with Mann - Whitney U - test and the p - values show the significant difference . The data represent at least 235 cells per condition from three independent experiments .

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(A) Left: Representative images of S9.6 immunofluorescence of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of S9.6 average nuclear intensity of U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) after transfection with either an empty plasmid or a plasmid expressing DDX5. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference. The data represent at least 235 cells per condition from three independent experiments.


( B ) Top : Representative images of in situ PLA performed with anti - DDX5 and S9 . 6 antibodies in EdU - labelled U2OS cells . Where indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . Bottom : Quantification of PLA spots per nucleus in each condition as indicated . At least 300 cells per condition were counted from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test , the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

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(B) Top: Representative images of in situ PLA performed with anti-DDX5 and S9.6 antibodies in EdU-labelled U2OS cells. Where indicated, cells were transfected with a plasmid expressing RNase H1 (RH). Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Bottom: Quantification of PLA spots per nucleus in each condition as indicated. At least 300 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test, the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( C ) Representative screenshot of a specific genomic region showing DRIPc - seq profiles at Watson ( W ) and Crick ( C ) strands in K562 cells depleted of DDX5 ( siDDX5 ) or control cells ( siC ) from two independent experiments .

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(C) Representative screenshot of a specific genomic region showing DRIPc-seq profiles at Watson (W) and Crick (C) strands in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) from two independent experiments.


( D ) DNA - RNA hybrid distribution along protein - coding genes containing DRIPc - seq peaks in both conditions ( siC and siDDX5 ) and replicates . Gene metaplots represent the mean of antisense or sense mean DRIPc - seq signal from two independent experiments in K562 cells depleted of DDX5 ( siDDX5 ) or control cells ( siC ) as indicated .

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(D) DNA-RNA hybrid distribution along protein-coding genes containing DRIPc-seq peaks in both conditions (siC and siDDX5) and replicates. Gene metaplots represent the mean of antisense or sense mean DRIPc-seq signal from two independent experiments in K562 cells depleted of DDX5 (siDDX5) or control cells (siC) as indicated.


( A ) Left : Representative images of in situ PLA between S9 . 6 and γH2AX antibodies in U2OS cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) . When indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) 24h before or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls from non - irradiated siC cells are shown . Scale bar indicates 10 µm . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . Right : Quantification of PLA spots per nucleus in each condition as indicated . At least 500 cells per condition were counted from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

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(A) Left: Representative images of in situ PLA between S9.6 and γH2AX antibodies in U2OS cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC). When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 24h before or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls from non-irradiated siC cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Right: Quantification of PLA spots per nucleus in each condition as indicated. At least 500 cells per condition were counted from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( B ) DRIP - qPCR signal values at RBMXL1 and HIST1H2BG loci in U2OS DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1 ( RH ) pre - immunoprecipitation where indicated . The experiment was performed in both untreated cells ( - OHT ) and after tamoxifen addition ( + OHT ) . The data represent the mean ± SEM from at least four independent experiments . The statistical significance of the difference was calculated with unpaired one - tail t - test and the p - values show the significant difference .

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(B) DRIP-qPCR signal values at RBMXL1 and HIST1H2BG loci in U2OS DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1 (RH) pre-immunoprecipitation where indicated. The experiment was performed in both untreated cells (-OHT) and after tamoxifen addition (+OHT). The data represent the mean ± SEM from at least four independent experiments. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant difference.


( A ) Top : Scheme showing the experimental set up for laser irradiation in DDX5 - GFP transfected U2OS cells depleted of BRCA2 ( siBRCA2 ) or control cells ( siC ) . Middle : Live cell imaging of the recruitment of GFP - 53BP1 or DDX5 - GFP to DNA damage tracks at different time points as indicated . Exposure and processing were adjusted to best demonstrate stripes and anti - stripes . Scale bar indicates 10 µm . Bottom left : Western blot showing the siRNA mediated knock - down of BRCA2 from U2OS cells transfected with DDX5 - GFP . Bottom right : Quantification of the percentage of transfected cells that exhibit DDX5 - GFP " anti - stripe " pattern ( reduced GFP signal at DNA damage tracks compared to the signal in the nucleus ) at the times indicated in cells depleted of BRCA2 ( siBRCA2 ) or treated with control siRNA ( siC ) . The data represent the mean ± SEM from three independent experiments .

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(A) Top: Scheme showing the experimental set up for laser irradiation in DDX5-GFP transfected U2OS cells depleted of BRCA2 (siBRCA2) or control cells (siC). Middle: Live cell imaging of the recruitment of GFP-53BP1 or DDX5-GFP to DNA damage tracks at different time points as indicated. Exposure and processing were adjusted to best demonstrate stripes and anti-stripes. Scale bar indicates 10 µm. Bottom left: Western blot showing the siRNA mediated knock-down of BRCA2 from U2OS cells transfected with DDX5-GFP. Bottom right: Quantification of the percentage of transfected cells that exhibit DDX5-GFP "anti-stripe" pattern (reduced GFP signal at DNA damage tracks compared to the signal in the nucleus) at the times indicated in cells depleted of BRCA2 (siBRCA2) or treated with control siRNA (siC). The data represent the mean ± SEM from three independent experiments.


( B ) DDX5 ChIP - qPCR signal values at RBMXL1 and ASXL1 loci in U2OS DIvA cells transfected with the indicated siRNAs and either untreated cells ( - OHT ) or after tamoxifen addition ( + OHT ) . The data represent the mean ± SEM from three independent experiments . The green line represents the background levels of DDX5 signal . The statistical significance of the difference was calculated with unpaired one - tail t - test and the p - values show the significant differences between untreated cells ( - OHT ) and after tamoxifen addition ( + OHT ) .

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(B) DDX5 ChIP-qPCR signal values at RBMXL1 and ASXL1 loci in U2OS DIvA cells transfected with the indicated siRNAs and either untreated cells (-OHT) or after tamoxifen addition (+OHT). The data represent the mean ± SEM from three independent experiments. The green line represents the background levels of DDX5 signal. The statistical significance of the difference was calculated with unpaired one-tail t-test and the p-values show the significant differences between untreated cells (-OHT) and after tamoxifen addition (+OHT).


( C ) Top : Representative images of immunofluorescence of U2OS DIvA cells depleted of BRCA2 ( siBRCA2 ) , DDX5 ( siDDX5 ) or control cells ( siC ) and either untreated cells ( - OHT ) or after tamoxifen addition ( + OHT ) , as indicated . Scale bar indicates 10 µm . Bottom : Quantification of the number of γH2AX foci per nucleus ( left ) and DDX5 nuclear intensity ( right ) . The data represent at least 800 cells per condition from three independent experiments . The red line in the plot indicates the median and each symbol represents the value of a single cell . The statistical significance of the difference was calculated with Mann - Whitney U - test and the p - values show the significant difference .

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(C) Top: Representative images of immunofluorescence of U2OS DIvA cells depleted of BRCA2 (siBRCA2), DDX5 (siDDX5) or control cells (siC) and either untreated cells (-OHT) or after tamoxifen addition (+OHT), as indicated. Scale bar indicates 10 µm. Bottom: Quantification of the number of γH2AX foci per nucleus (left) and DDX5 nuclear intensity (right). The data represent at least 800 cells per condition from three independent experiments. The red line in the plot indicates the median and each symbol represents the value of a single cell. The statistical significance of the difference was calculated with Mann-Whitney U-test and the p-values show the significant difference.


( A ) Top : PAGE gel showing a representative unwinding assays in which purified MBP - DDX5 - GST ( 1 - 5 nM ) was incubated with 32P - labelled synthetic R - loop substrate in presence or absence of 2 nM purified EGFP - MBP - BRCA2 ( top gel left ) or 10 nM purified BRCA2LT3 ( top gel right ) or 50 nM purified BRCA2T1 ( bottom gel ) . Bottom : Quantification of the unwinding experiments showing the percentage of free RNA relative to the R - loop substrate ( unwound product ) as a function of DDX5 concentration alone ( black ) or in presence of BRCA2 ( red ) or BRCA2LT3 ( blue ) or BRCA2T1 ( pink ) . The data represent the mean ± SD of at least three independent experiments . Right : SDS - PAGE showing 500 ng of purified 2XMBP - BRCA2LT3 and 150 ng of purified BRCA2 used in the unwinding assay .

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(A) Top: PAGE gel showing a representative unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with 32P-labelled synthetic R-loop substrate in presence or absence of 2 nM purified EGFP-MBP-BRCA2 (top gel left) or 10 nM purified BRCA2LT3 (top gel right) or 50 nM purified BRCA2T1 (bottom gel). Bottom: Quantification of the unwinding experiments showing the percentage of free RNA relative to the R-loop substrate (unwound product) as a function of DDX5 concentration alone (black) or in presence of BRCA2 (red) or BRCA2LT3 (blue) or BRCA2T1 (pink). The data represent the mean ± SD of at least three independent experiments. Right: SDS-PAGE showing 500 ng of purified 2XMBP-BRCA2LT3 and 150 ng of purified BRCA2 used in the unwinding assay.


( B ) Left : Thin layer chromatography ( TLC ) plate showing a representative ATP hydrolysis assay in which purified MBP - DDX5 - GST ( 50 - 200 nM ) was incubated with R - loop synthetic substrate and [ γ32P ] ATP in presence or absence of or 6 nM purified BRCA2LT3 . Right : Quantification of the ATP hydrolysed in each condition . No protein control was used as background . The data represent the mean ± SD from three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O 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-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O 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) Left: Thin layer chromatography (TLC) plate showing a representative ATP hydrolysis assay in which purified MBP-DDX5-GST (50-200 nM) was incubated with R-loop synthetic substrate and [γ32P] ATP in presence or absence of or 6 nM purified BRCA2LT3. Right: Quantification of the ATP hydrolysed in each condition. No protein control was used as background. The data represent the mean ± SD from three independent experiments.


( A ) Left : GFP pull - down assay from whole cells extracts of BRCA2 deficient DLD1 expressing BRCA2 ( WT ) or the variant T207A ( T207A ) . DLD1 BRCA2 + / + ( + / + ) cell extracts were used as control for the GFP - trap . DDX5 and BRCA2 were detected with specific antibodies against DDX5 and BRCA2 , respectively . StainFree images of the gels before transfer was used as loading control ( cropped image is shown ) . Right : Quantification of the GFP - trap pull - down experiments calculated as the co - immunoprecipitated DDX5 with either BRCA2 WT or BRCA2 - T207A relative to the input levels of DDX5 and the amount of immunoprecipitated EGFPMBP - BRCA2 . Results are presented as the fold change compared to the BRCA2 WT clone . The data represents the mean ± SD of three independent experiments . Statistical significance of the difference was calculated with unpaired t test and the p - values show the significant difference .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Left: GFP pull-down assay from whole cells extracts of BRCA2 deficient DLD1 expressing BRCA2 (WT) or the variant T207A (T207A). DLD1 BRCA2+/+ (+/+) cell extracts were used as control for the GFP-trap. DDX5 and BRCA2 were detected with specific antibodies against DDX5 and BRCA2, respectively. StainFree images of the gels before transfer was used as loading control (cropped image is shown). Right: Quantification of the GFP-trap pull-down experiments calculated as the co-immunoprecipitated DDX5 with either BRCA2 WT or BRCA2-T207A relative to the input levels of DDX5 and the amount of immunoprecipitated EGFPMBP-BRCA2. Results are presented as the fold change compared to the BRCA2 WT clone. The data represents the mean ± SD of three independent experiments. Statistical significance of the difference was calculated with unpaired t test and the p-values show the significant difference.


( B ) Left : Representative images of S9 . 6 immunofluorescence of DLD1 cells bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . The merged images show the signal of S9 . 6 , nucleolin ( nucleoli ) antibodies and DAPI staining . Scale bar indicates 25 µm . Right : Quantification of the average nuclear intensity of S9 . 6 antibody in DLD1 BRCA2 - WT ( WT ) or BRCA2 - T207A ( T207A ) cells transfected with either a plasmid expressing GFP alone ( - RH ) or GFP - RNase H1 ( + RH ) , as indicated . The red line in the plot indicates the median and each symbol represents the value of a single cell . Data correspond to at least 170 cells per condition from two independent experiments . The statistical significance of the difference was calculated with Mann - Whitney U - test ; p - values show the significant difference .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Representative images of S9.6 immunofluorescence of DLD1 cells bearing BRCA2 (WT) or BRCA2-T207A (T207A). The merged images show the signal of S9.6, nucleolin (nucleoli) antibodies and DAPI staining. Scale bar indicates 25 µm. Right: Quantification of the average nuclear intensity of S9.6 antibody in DLD1 BRCA2-WT (WT) or BRCA2-T207A (T207A) cells transfected with either a plasmid expressing GFP alone (-RH) or GFP-RNase H1 (+RH), as indicated. The red line in the plot indicates the median and each symbol represents the value of a single cell. Data correspond to at least 170 cells per condition from two independent experiments. The statistical significance of the difference was calculated with Mann-Whitney U-test; p-values show the significant difference.


( C ) Relative DRIP - qPCR signal values ( respect to the siC level at each locus ) at the MALAT1 , RRPH1 and HIST1H2BG loci in DLD1 BRCA2 - / - cells bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) and treated in vitro with RNase H1 ( RH ) pre - immunoprecipitation where indicated . The data represent the mean ± SEM from seven independent experiments . The statistical significance of the difference was calculated with paired t - Student test ; the p - values show the significant difference .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Relative DRIP-qPCR signal values (respect to the siC level at each locus) at the MALAT1, RRPH1 and HIST1H2BG loci in DLD1 BRCA2-/- cells bearing BRCA2 (WT) or BRCA2-T207A (T207A) and treated in vitro with RNase H1 (RH) pre-immunoprecipitation where indicated. The data represent the mean ± SEM from seven independent experiments. The statistical significance of the difference was calculated with paired t-Student test; the p-values show the significant difference.


( D ) Top : Representative images of In situ PLA performed between BRCA2 and DDX5 antibodies in cells bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) , as indicated . Cells were fixed directly or 4h post - irradiation ( 6Gy ) . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Bottom : Quantification of the number of PLA spots per nucleus . At least 750 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Top: Representative images of In situ PLA performed between BRCA2 and DDX5 antibodies in cells bearing BRCA2 (WT) or BRCA2-T207A (T207A), as indicated. Cells were fixed directly or 4h post-irradiation (6Gy). Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 750 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( A ) Top : Representative images of In situ PLA performed between DDX5 and S9 . 6 ( DNA - RNA hybrids ) antibodies in BRCA2 deficient DLD1 cells ( BRCA2 - / - ) or DLD1 stable clones expressing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . Cells were fixed directly or 4h post - irradiation ( 6Gy ) . When indicated , cells were transfected / treated with RNase H1 ( RH ) prior to fixation . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Bottom : Quantification of the number of PLA spots per nucleus . At least 500 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot . ( B ) Top : Representative images of In situ PLA performed between DDX5 and γH2AX antibodies in BRCA2 deficient DLD1 cells ( BRCA2 - / - ) bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . Cells were fixed directly or 4h post - irradiation ( 6Gy ) . When indicated , cells were transfected / treated with RNase H1 ( RH ) prior to fixation . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Bottom : Quantification of the number of PLA spots per nucleus . At least 600 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Top: Representative images of In situ PLA performed between DDX5 and S9.6 (DNA-RNA hybrids) antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) or DLD1 stable clones expressing BRCA2 (WT) or BRCA2-T207A (T207A). Cells were fixed directly or 4h post-irradiation (6Gy). When indicated, cells were transfected/treated with RNase H1 (RH) prior to fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 500 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot. (B) Top: Representative images of In situ PLA performed between DDX5 and γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2 (WT) or BRCA2-T207A (T207A). Cells were fixed directly or 4h post-irradiation (6Gy). When indicated, cells were transfected/treated with RNase H1 (RH) prior to fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Bottom: Quantification of the number of PLA spots per nucleus. At least 600 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( C ) Left : Representative images of the recruitment of transient transfected GFP - MBP - BRCA2 ( WT and T207A ) to DNA damage tracks at 5 min post - irradiation in U2OS cells . γH2AX is used as a marker of DNA damage . Scale bar indicates 10 µm . Right : Quantification of the intensity of GFP signal at the laser tracks . At least 130 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the two samples we applied an unpaired t - test . The red line in the plot indicates the median and each symbol represents a single cell intensity value .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Left: Representative images of the recruitment of transient transfected GFP-MBP-BRCA2 (WT and T207A) to DNA damage tracks at 5 min post-irradiation in U2OS cells. γH2AX is used as a marker of DNA damage. Scale bar indicates 10 µm. Right: Quantification of the intensity of GFP signal at the laser tracks. At least 130 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the two samples we applied an unpaired t-test. The red line in the plot indicates the median and each symbol represents a single cell intensity value.


( D ) Top : Representative images of in situ PLA performed between S9 . 6 ( DNA - RNA hybrids ) and anti - γH2AX antibodies in BRCA2 deficient DLD1 cells ( BRCA2 - / - ) bearing BRCA2 ( WT ) or BRCA2 - T207A ( T207A ) . When indicated , cells were transfected / treated with RNase H1 ( RH ) or treated with Cordycepin ( Cordy ) for 2h at 37 ° C before fixation . Single antibody controls in non - irradiated BRCA2 WT cells are shown . Scale bar indicates 10 µm . Nuclei as defined by auto threshold plugin on the DAPI image ( Image J ) our outlined in yellow . Bottom : Quantification of the number of PLA spots per nucleus . At least 600 cells were counted per condition from three independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single PLA spot .

O 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-MEASURED_VAR O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Top: Representative images of in situ PLA performed between S9.6 (DNA-RNA hybrids) and anti-γH2AX antibodies in BRCA2 deficient DLD1 cells (BRCA2-/-) bearing BRCA2 (WT) or BRCA2-T207A (T207A). When indicated, cells were transfected/treated with RNase H1 (RH) or treated with Cordycepin (Cordy) for 2h at 37°C before fixation. Single antibody controls in non-irradiated BRCA2 WT cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (Image J) our outlined in yellow. Bottom: Quantification of the number of PLA spots per nucleus. At least 600 cells were counted per condition from three independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single PLA spot.


( E ) Top : PAGE gel showing a representative unwinding assays in which purified MBP - DDX5 - GST ( 1 - 5 nM ) was incubated with 32P - labelled synthetic R - loop substrate in presence or absence of 50 nM purified BRCA2T1 - T207A ( bottom right ) . Bottom left : Quantification of the unwinding experiments showing the percentage of free RNA relative to the R - loop substrate ( unwound product ) as a function of DDX5 concentration alone ( black ) or in presence of BRCA2T1 - T207A ( green ) . The data represent the mean ± SD of at least three independent experiments . Bottom right : SDS - PAGE showing 650 ng of purified 2XMBP - BRCA2T1 - T207A used in the unwinding assay .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O 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-CONTROLLED_VAR O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O 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-MEASURED_VAR O O O O O O O O

(E) Top: PAGE gel showing a representative unwinding assays in which purified MBP-DDX5-GST (1-5 nM) was incubated with 32P-labelled synthetic R-loop substrate in presence or absence of 50 nM purified BRCA2T1-T207A (bottom right). Bottom left: Quantification of the unwinding experiments showing the percentage of free RNA relative to the R-loop substrate (unwound product) as a function of DDX5 concentration alone (black) or in presence of BRCA2T1-T207A (green). The data represent the mean ± SD of at least three independent experiments. Bottom right: SDS-PAGE showing 650 ng of purified 2XMBP-BRCA2T1-T207A used in the unwinding assay.


( A ) Top : Representative immunofluorescence images of cells stained for RAD51 ( green ) in U2OS cells depleted of DDX5 ( siDDX5 ) and in control cells ( siC ) in non - treated ( - ) or different time points after exposure to IR ( 6Gy ) , as indicated . Scale bar indicates 10 µm . Bottom : Graph showing the average number of RAD51 repair foci in both cell lines . The data represent the mean ± SEM of three independent experiments .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O

(A) Top: Representative immunofluorescence images of cells stained for RAD51 (green) in U2OS cells depleted of DDX5 (siDDX5) and in control cells (siC) in non-treated (-) or different time points after exposure to IR (6Gy), as indicated. Scale bar indicates 10 µm. Bottom: Graph showing the average number of RAD51 repair foci in both cell lines. The data represent the mean ± SEM of three independent experiments.


( B ) Top : Representative immunofluorescence images of DLD1 BRCA2 deficient cells ( BRCA2 - / - ) or BRCA2 - / - cells expressing BRCA2 WT ( WT ) or BRCA2 - T207A ( T207A ) in non - treated or at different time points post - IR ( 6 Gy ) , as indicated ; hybridized with anti - RAD51 antibody ( green ) . Scale bar indicates 10 µm . Bottom : Graph showing the average number of RAD51 repair foci in the three cell lines . The data represent the mean ± SEM of three independent experiments .

O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

(B) Top: Representative immunofluorescence images of DLD1 BRCA2 deficient cells (BRCA2-/-) or BRCA2-/- cells expressing BRCA2 WT (WT) or BRCA2-T207A (T207A) in non-treated or at different time points post-IR (6 Gy), as indicated; hybridized with anti-RAD51 antibody (green). Scale bar indicates 10 µm. Bottom: Graph showing the average number of RAD51 repair foci in the three cell lines. The data represent the mean ± SEM of three independent experiments.


( C ) Left : Representative immunofluorescence images of DLD1 BRCA2 WT or BRCA2 - T207A cells 4h post - irradiation ( 6Gy ) hybridized with anti - γH2AX and anti - RPA antibodies . Right : Quantification of the number of γH2AX and RPA foci per nucleus , as indicated . The data represent at least 400 cells per condition from two independent experiments . For statistical comparison of the differences between the samples we applied a Kruskal - Wallis test followed by Dunn ' s multiple comparison test and the p - values show significant differences . The red line in the plot indicates the median and each symbol represents a single focus .

O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Left: Representative immunofluorescence images of DLD1 BRCA2 WT or BRCA2-T207A cells 4h post-irradiation (6Gy) hybridized with anti-γH2AX and anti-RPA antibodies. Right: Quantification of the number of γH2AX and RPA foci per nucleus, as indicated. The data represent at least 400 cells per condition from two independent experiments. For statistical comparison of the differences between the samples we applied a Kruskal-Wallis test followed by Dunn's multiple comparison test and the p-values show significant differences. The red line in the plot indicates the median and each symbol represents a single focus.