Figure 2 ( 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 . ( 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 .

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

Figure 2(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.(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.


Figure 3 ( 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 .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


Figure 4 ( 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 ) . ( 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 .

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

Figure 4(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).(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.


Figure 5 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(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.


Figure 6 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6(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.(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.


Figure 7 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.(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.


Figure 8 ( 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 . ( 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 . ( D ) Left : Representative immunofluorescence images of DLD1 BRCA2 - T207A cells 2h post - irradiation ( 6Gy ) , as indicated , hybridized with anti - γH2AX and anti - RAD51 antibodies . ( RH ) RNase H . Right : Quantification of the number of γH2AX foci ( left ) or RAD51 foci ( right ) per nucleus . When indicated , cells were transfected with a plasmid expressing RNase H1 ( RH ) 48h prior fixation . The data shown is from at least 400 cells 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 focus . ( E ) Top : Subcellular fractionation showing amount of RAD51 in DLD1 stable clones expressing BRCA2 - T207A ( T207A ) in chromatin fraction . When indicated , cells were irradiated ( 2h treatment ) and / or transfected with a plasmid expressing RNase H1 ( RH ) 48h prior fixation . α - tubulin and Histone H3 signals are shown as markers of cytoplasmic and chromatin fraction respectively . Bottom : quantification of RAD51 levels detected by Western blot with a RAD51 specific antibody in chromatin fraction relative to RAD51 levels in WCE . Results are presented as the fold change compared to the untreated sample . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8(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.(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.(D) Left: Representative immunofluorescence images of DLD1 BRCA2-T207A cells 2h post-irradiation (6Gy), as indicated, hybridized with anti-γH2AX and anti-RAD51 antibodies. (RH) RNase H. Right: Quantification of the number of γH2AX foci (left) or RAD51 foci (right) per nucleus. When indicated, cells were transfected with a plasmid expressing RNase H1 (RH) 48h prior fixation. The data shown is from at least 400 cells 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 focus.(E) Top: Subcellular fractionation showing amount of RAD51 in DLD1 stable clones expressing BRCA2-T207A (T207A) in chromatin fraction. When indicated, cells were irradiated (2h treatment) and/or transfected with a plasmid expressing RNase H1 (RH) 48h prior fixation. α-tubulin and Histone H3 signals are shown as markers of cytoplasmic and chromatin fraction respectively. Bottom: quantification of RAD51 levels detected by Western blot with a RAD51 specific antibody in chromatin fraction relative to RAD51 levels in WCE. Results are presented as the fold change compared to the untreated sample. 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.


Figure 1Cell viability analysis using CellTiter - Glo of BN175 cells treated with the indicated agents for 24 hrs and 48 hrs ( n = 4 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . DMSO ( Unt ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) . SM represents SM - 164 . Error bars represent SD and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 . TNF - induced complex - II immuno - precipitation . BN175 cells were treated with the indicated agents for 8 hrs . FADD immuno - precipitation was performed followed by western blot analysis ( n = 3 biological replicates , shown is a representative experiment ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) . Proximity ligation assay between Ripk1 and Casp - 8 performed in BN175 cells upon treatment with the indicated agents for 8 hrs ( n = 3 biological replicates , shown are representative images ) . Scale bar represents 10 μm . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) . DEVDase activity assay of BN175 cells treated with the indicated drugs for 6 hrs ( n = 3 biological replicates ) . DMSO ( Unt ) , TNF ( 10 ng / ml ) , SM ( 100 ng / ml ) and Mel ( 3 . 3 µM ) . Error bars represent SD and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 . Cell viability analysis using CellTiter - Glo of BN175 CRISPR / Cas9 Ripk1 and Casp - 8 knockouts ( KO ) cells treated with the indicated agents for 18 and 48 hrs ( n = 3 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR / Cas9 control ( Ctrl ) , * * * * P ≤ 0 . 0001 .

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

Figure 1Cell viability analysis using CellTiter-Glo of BN175 cells treated with the indicated agents for 24 hrs and 48 hrs (n = 4 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). SM represents SM-164. Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.TNF-induced complex-II immuno-precipitation. BN175 cells were treated with the indicated agents for 8 hrs. FADD immuno-precipitation was performed followed by western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).Proximity ligation assay between Ripk1 and Casp-8 performed in BN175 cells upon treatment with the indicated agents for 8 hrs (n = 3 biological replicates, shown are representative images). Scale bar represents 10 μm. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM).DEVDase activity assay of BN175 cells treated with the indicated drugs for 6 hrs (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml), SM (100 ng/ml) and Mel (3.3 µM). Error bars represent SD and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 knockouts (KO) cells treated with the indicated agents for 18 and 48 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001.


Figure 2TNF - induced complex - II immuno - precipitation . HT1080 cells were treated with the indicated agents for 8 hrs . CASP - 8 immuno - precipitation was performed followed by western blot analysis ( n = 3 biological replicates , shown is a representative experiment ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . Cell death analysis measured by Celigo of PI - positive HT1080 cells , cells were treated with the indicated agents for 24 hrs . ( n = 3 biological replicates ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD and an unpaired t - test was performed , * * * * P ≤ 0 . 0001 . Cell death analysis by Celigo of PI - positive HT1080 cells treated with the indicated agents for 72 hrs following siCTRL , siRIPK1 or siCASP - 8 knockdown for 48 hrs ( n = 3 biological replicates ) . DMSO ( Unt ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed , * * * * P ≤ 0 . 0001 . TNF - induced complex - II immuno - precipitation . A375 cells were treated with the indicated agents for 6 hrs . Caspase - 8 immuno - precipitation was performed followed by western blot analysis . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) ( n = 3 biological replicates , shown is a representative experiment ) . Cell death analysis by Celigo of PI - positive A375 treated with the indicated agents for 12 hrs . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 . Cell viability analysis using CellTiter - Glo of A375 cells treated with the indicated drugs for 24 hrs following siCTRL , siRIPK1 or siCASP - 8 knockdown for 48hrs ( n = 3 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . DMSO ( Unt ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed to compare the mean value of each treatment to the treated A375 siCTRL , * * * * P ≤ 0 . 0001 . Cell death analysis by Celigo of PI - positive MeWo ( G ) , DO4 ( H ) or SW - 872 ( I ) cells . Cells were treated with the indicated agents for 24 hrs ( MeWO and SW - 872 ) or 12 hrs ( DO4 ) . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * * P ≤ 0 . 0001 . Cell death analysis by Celigo of PI - positive HS - ES - 2R Cells were treated with the indicated agents for 48 hrs ( HS - ES - 2R ) DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 100 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * P ≤ 0 . 001 . Cell death analysis by Celigo of PI - positive HS - SY - II cells . Cells were treated with the indicated agents for 24 hrs ( HS - SY - II ) . DMSO ( Unt ) , TNF ( 100 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 3 , and statistical analysis was performed with an unpaired t - test , * * * P ≤ 0 . 001 . Cell Death analysis by IncuCyte Zoom of CellTox Green - positive MET1 ( L ) , MET2 ( M ) and IC8 ( N ) cells . Cells were treated with the indicated agents for 40 hrs . DMSO ( Unt ) , zVAD ( 10 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) SM represents SM - 164 . Error bars represent SD . Displayed are representative results from n = 6 , and statistical analysis was performed with a one - way Anova , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 .

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

Figure 2TNF-induced complex-II immuno-precipitation. HT1080 cells were treated with the indicated agents for 8 hrs. CASP-8 immuno-precipitation was performed followed by western blot analysis (n = 3 biological replicates, shown is a representative experiment). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml).Cell death analysis measured by Celigo of PI-positive HT1080 cells, cells were treated with the indicated agents for 24 hrs. (n = 3 biological replicates). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and an unpaired t-test was performed, **** P ≤ 0.0001.Cell death analysis by Celigo of PI-positive HT1080 cells treated with the indicated agents for 72 hrs following siCTRL, siRIPK1 or siCASP-8 knockdown for 48 hrs (n = 3 biological replicates). DMSO (Unt), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed, **** P ≤ 0.0001.TNF-induced complex-II immuno-precipitation. A375 cells were treated with the indicated agents for 6 hrs. Caspase-8 immuno-precipitation was performed followed by western blot analysis. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) (n = 3 biological replicates, shown is a representative experiment).Cell death analysis by Celigo of PI-positive A375 treated with the indicated agents for 12 hrs. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.Cell viability analysis using CellTiter-Glo of A375 cells treated with the indicated drugs for 24 hrs following siCTRL, siRIPK1 or siCASP-8 knockdown for 48hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated A375 siCTRL, **** P ≤ 0.0001.Cell death analysis by Celigo of PI-positive MeWo (G), DO4 (H) or SW-872 (I) cells. Cells were treated with the indicated agents for 24 hrs (MeWO and SW-872) or 12 hrs (DO4). DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, **** P ≤ 0.0001.Cell death analysis by Celigo of PI-positive HS-ES-2R Cells were treated with the indicated agents for 48 hrs (HS-ES-2R) DMSO (Unt), zVAD (10 µM), TNF (100 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, *** P ≤ 0.001.Cell death analysis by Celigo of PI-positive HS-SY-II cells. Cells were treated with the indicated agents for 24 hrs (HS-SY-II). DMSO (Unt), TNF (100 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=3, and statistical analysis was performed with an unpaired t-test, *** P ≤ 0.001.Cell Death analysis by IncuCyte Zoom of CellTox Green-positive MET1 (L), MET2 (M) and IC8 (N) cells. Cells were treated with the indicated agents for 40 hrs. DMSO (Unt), zVAD (10 µM), TNF (10 ng/ml) and SM (100 ng/ml) SM represents SM-164. Error bars represent SD. Displayed are representative results from n=6, and statistical analysis was performed with a one-way Anova, *** P ≤ 0.001, **** P ≤ 0.0001.


Figure 3Measurements of tumour volume of the respective cohorts ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . The addition of SM to the standard - of - care treatment ILP - TNF / Mel significantly prolonged survival compared to either modality alone ( n = 6 rats per cohort ) . Statistical analysis was performed with a log - rank test , * * P ≤ 0 . 01 . Median TNF / Mel is 0 . 4472 and TNF / Mel / SM is 1 . 154 . TNF / Mel 25th percentile limit 0 . 02887 and 75th percentile limit 0 . 7627 . TNF / Mel / SM 25th percentile limit 1 . 136 and 75th percentile limit 1 . 67 . TNF / Mel Min error bar - 0 . 2389 and Max 0 . 7687 . TNF / SM / Mel Min error bar 1 . 12 and Max 1 . 813 . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * * P ≤ 0 . 01 . DEVDase activity assay of parental BN175 cells and drug - tolerant tumour cell lines isolated from ILP - TNF / Mel / SM treated animals ( 124 , 133 , 136 ) , treated with the indicated drugs for 6 hrs ( n = 4 biological repeats ) . DMSO ( Unt ) , Mel ( 3 . 3 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . Cell viability analysis using CellTiter - Glo of BN175 parental and 124 , 133 and 136 cells treated with the indicated drugs for 24 hrs ( n = 3 biological repeats ) . DMSO ( Unt ) , Mel ( 3 . 3 µM ) , TNF ( 10 ng / ml ) and SM ( 100 ng / ml ) . SM represents SM - 164 . Error bars represent SD . Statistical analysis was performed with a one - way Anova , ns = not significant . Cell viability analysis using CellTiter - Glo of BN175 CRISPR / Cas9 Ripk1 and Casp - 8 KO cells treated with the indicated agents for 24 hrs ( n = 3 biological replicates ) . Cell viability values are displayed as a percentage of the relative untreated control . DMSO ( Unt ) , SM ( 100 ng / ml ) and Riboxxol ( 1 µg / ml ) . SM represents SM - 164 . Error bars represent SD and a one - way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR / Cas9 control ( Ctrl ) , * * * * P ≤ 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, * P ≤ 0.05, *** P ≤ 0.001.The addition of SM to the standard-of-care treatment ILP-TNF/Mel significantly prolonged survival compared to either modality alone (n = 6 rats per cohort). Statistical analysis was performed with a log-rank test, ** P ≤ 0.01.Median TNF/Mel is 0.4472 and TNF/Mel/SM is 1.154. TNF/Mel 25th percentile limit 0.02887 and 75th percentile limit 0.7627. TNF/Mel/SM 25th percentile limit 1.136 and 75th percentile limit 1.67. TNF/Mel Min error bar -0.2389 and Max 0.7687. TNF/SM/Mel Min error bar 1.12 and Max 1.813. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ** P ≤ 0.01.DEVDase activity assay of parental BN175 cells and drug-tolerant tumour cell lines isolated from ILP-TNF/Mel/SM treated animals (124, 133, 136), treated with the indicated drugs for 6 hrs (n = 4 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml).Cell viability analysis using CellTiter-Glo of BN175 parental and 124, 133 and 136 cells treated with the indicated drugs for 24 hrs (n = 3 biological repeats). DMSO (Unt), Mel (3.3 µM), TNF (10 ng/ml) and SM (100 ng/ml). SM represents SM-164. Error bars represent SD. Statistical analysis was performed with a one-way Anova, ns=not significant.Cell viability analysis using CellTiter-Glo of BN175 CRISPR/Cas9 Ripk1 and Casp-8 KO cells treated with the indicated agents for 24 hrs (n = 3 biological replicates). Cell viability values are displayed as a percentage of the relative untreated control. DMSO (Unt), SM (100 ng/ml) and Riboxxol (1 µg/ml). SM represents SM-164. Error bars represent SD and a one-way Anova was performed to compare the mean value of each treatment to the treated BN175 CRISPR/Cas9 control (Ctrl), **** P ≤ 0.0001.


Figure 4Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . ILP with TNF / Mel / SM significantly increased infiltration by CD3 + lymphocytes ( B ) and CD3 - CD161 + NK cells ( C ) . Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . While ILP treatment with TNF / Mel significantly reduced Granzyme B positive NK cells ( D ) , the numbers of Granzyme B positive NK cells were restored to normal levels upon co - treatment with SM ( TNF / Mel / SM ) . Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . Importantly , ILP - mediated treatment with TNF / Mel / SM significantly increased the numbers of CD3 + CD8 + cytotoxic T cells ( E ) . Flow cytometry analysis of tumours collected on Day 10 following treatment ( n = 3 animals per group ) . ILP - TNF / Mel / SM significantly upregulated Granzyme B in CD3 + CD8 + CD161 - cytoxtoxic T cellsILP - TNF / Mel / SM significantly reduced infiltration by CD3 + CD4 + Foxp3 - T cells whilst ( G ) increasing infiltrations by CD3 + CD4 + Foxp3 + T regulatory cells ( H ) . A non - significant increase in T regulatory cell ICOS expression was also noted ( I ) .

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Figure 4Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP with TNF/Mel/SM significantly increased infiltration by CD3+ lymphocytes (B) and CD3-CD161+ NK cells (C).Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). While ILP treatment with TNF/Mel significantly reduced Granzyme B positive NK cells (D), the numbers of Granzyme B positive NK cells were restored to normal levels upon co-treatment with SM (TNF/Mel/SM).Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). Importantly, ILP-mediated treatment with TNF/Mel/SM significantly increased the numbers of CD3+CD8+ cytotoxic T cells (E).Flow cytometry analysis of tumours collected on Day 10 following treatment (n = 3 animals per group). ILP-TNF/Mel/SM significantly upregulated Granzyme B in CD3+CD8+CD161- cytoxtoxic T cellsILP-TNF/Mel/SM significantly reduced infiltration by CD3+CD4+Foxp3- T cells whilst (G) increasing infiltrations by CD3+CD4+Foxp3+ T regulatory cells (H).A non-significant increase in T regulatory cell ICOS expression was also noted (I).


Figure 5Measurements of tumour volume of the respective cohorts ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD . The addition of either anti - CTLA - 4 or anti - PD - 1 antibodies prolonged survival in comparison to ILP - TNF / Mel / SM alone ( n = 6 animals per cohort ) . SM represents Birinapant . Statistical analysis was performed with a log - rank test , * P ≤ 0 . 05 . Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry ( n = 3 per cohort ) . The addition of anti - CTLA - 4 antibodies led to a significant increase in CD4 + Foxp3 - helper T cells ( C ) accompanied by a depletion of CD4 + Foxp3 + regulatory T cells ( D ) . SM represents Birinapant . Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry ( n = 3 per cohort ) . The addition of anti - PD - 1 antibodies led to an increase in Granzyme B ( GzmB ) expression on NK cells ( E ) . SM represents Birinapant .

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Figure 5Measurements of tumour volume of the respective cohorts (n = 6 animals per cohort). SM represents Birinapant. Error bars represent SD.The addition of either anti-CTLA-4 or anti-PD-1 antibodies prolonged survival in comparison to ILP-TNF/Mel/SM alone (n = 6 animals per cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05.Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-CTLA-4 antibodies led to a significant increase in CD4+ Foxp3- helper T cells (C) accompanied by a depletion of CD4+ Foxp3+ regulatory T cells (D). SM represents Birinapant.Tumours were collected 4 days after the ILP procedure and analysed by flow cytometry (n = 3 per cohort). The addition of anti-PD-1 antibodies led to an increase in Granzyme B (GzmB) expression on NK cells (E). SM represents Birinapant.


Figure 6Tumour volumes at the time of surgery following ILP - TNF / Mel or ILP - TNF / Mel / SM ( n = 6 animals per cohort ) . SM represents Birinapant . Error bars represent SD . Statistical analysis was performed with an unpaired t - test , * * * * , P ≤ 0 . 0001 . Individual growth curves following surgery indicated improved control following ILP - TNF / Mel / SM ( n = 6 animals per cohort ) . Tumour volume on re - challenge with BN175 sarcoma on contralateral limb of long - term survivors following ILP - TNF / Mel or ILP - TNF / Mel / SM ( n = 3 animals per TNF / Mel cohort and n = 5 per TNF / Mel / SM cohort ) . Unless otherwise stated , SM represents Birinapant . Error bars represent S . D . ILP - TNF / Mel / SM significantly delayed growth leading to prolonged survival on re - challenge ( n = 3 animals per TNF / Mel cohort and n = 5 per TNF / Mel / SM cohort ) . SM represents Birinapant . Statistical analysis was performed with a log - rank test , * P ≤ 0 . 05 .

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

Figure 6Tumour volumes at the time of surgery following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=6 animals per cohort). SM represents Birinapant. Error bars represent SD. Statistical analysis was performed with an unpaired t-test, ****, P ≤ 0.0001.Individual growth curves following surgery indicated improved control following ILP-TNF/Mel/SM (n=6 animals per cohort).Tumour volume on re-challenge with BN175 sarcoma on contralateral limb of long-term survivors following ILP-TNF/Mel or ILP-TNF/Mel/SM (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). Unless otherwise stated, SM represents Birinapant. Error bars represent S.D.ILP-TNF/Mel/SM significantly delayed growth leading to prolonged survival on re-challenge (n=3 animals per TNF/Mel cohort and n=5 per TNF/Mel/SM cohort). SM represents Birinapant. Statistical analysis was performed with a log-rank test, * P ≤ 0.05.


Figure 1 : Westernblot of producer cell lysates and concentrated reporter particles . BHK cells were transfected with either human ACE2 or empty vector , subsequently infected with VSV - reporter particles pseudotyped with chimeric spikes , luciferase was measured and normalized to no pseudotype as a readout for cell entry . Shown are the data for 3 replicates .

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Figure 1:Westernblot of producer cell lysates and concentrated reporter particles.BHK cells were transfected with either human ACE2 or empty vector, subsequently infected with VSV-reporter particles pseudotyped with chimeric spikes, luciferase was measured and normalized to no pseudotype as a readout for cell entry. Shown are the data for 3 replicates.


Figure 2 : Primate cells , were infected with VSV - particles pseudotyped with the lineage B chimeric spike panel . Pseudotypes were either left untreated or incubated with trypsin before addition to the cells . Luciferase was measured and normalized to particles produced without pseudotype . Shown are the data for 3 replicates . human cells were infected with VSV - particles pseudotyped with the lineage B chimeric spike panel . Pseudotypes were either left untreated or incubated with trypsin before addition to the cells . Luciferase was measured and normalized to particles produced without pseudotype . Shown are the data for 3 replicates . bat cells were infected with VSV - particles pseudotyped with the lineage B chimeric spike panel . Pseudotypes were either left untreated or incubated with trypsin before addition to the cells . Luciferase was measured and normalized to particles produced without pseudotype . Shown are the data for 3 replicates .

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Figure 2:Primate cells, were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.human cells were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.bat cells were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.


Figure 3 : Pseudotyped particles were either left untreated and treated with trypsin and subsequently used to infect BHK cells transfected with the coronavirus receptor indicated . Shown are data from 3 replicates . Expression and pseudotype incorporation of SARS - S - 2019 - nCoV RBD chimeras . Pseudotypes were used to infect cells expressing hACE2 , hDPP4 , hAPN , or empty vector , without protease treatment .

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Figure 3:Pseudotyped particles were either left untreated and treated with trypsin and subsequently used to infect BHK cells transfected with the coronavirus receptor indicated. Shown are data from 3 replicates.Expression and pseudotype incorporation of SARS-S-2019-nCoV RBD chimeras.Pseudotypes were used to infect cells expressing hACE2, hDPP4, hAPN, or empty vector, without protease treatment.


Figure 4 : VSV pseudotypes were generated with the indicated RBD and used to infect BHKs transfected with either human ACE2 or empty vector . Shown are data for 3 replicates . Westernblot of producer cell lysates and concentrated pseudotyped particles .

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Figure 4:VSV pseudotypes were generated with the indicated RBD and used to infect BHKs transfected with either human ACE2 or empty vector. Shown are data for 3 replicates.Westernblot of producer cell lysates and concentrated pseudotyped particles.


Figure 5 : Westernblot of producer cell lysates and concentrated pseudotypes particles . Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect Huh - 7 . 5 cells . Shown are data for 3 replicates . Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect BHK cells transfected with human ACE2 . Shown are data for 3 replicates .

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Figure 5:Westernblot of producer cell lysates and concentrated pseudotypes particles.Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect Huh-7.5 cells. Shown are data for 3 replicates.Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect BHK cells transfected with human ACE2. Shown are data for 3 replicates.


Figure 1c , Log normalised expression of markers for epithelial ( EPCAM ) , mature luminal epithelial ( ESR1 ) , myoepithelial ( KRT5 , KRT14 and ACTA2 ) and proliferating cancer cells ( MKI67 ) . e , Log normalised expression of markers for fibroblasts ( PDGFRB , THY1 , COL1A1 , ITGB1 and S100A4 ) , endothelial cells ( PECAM1 ) , T - cells ( CD3D ) , CD8 T cells ( CD8A ) , T - regulatory cells ( FOXP3 ) , B - cells ( MS4A1 ) , myeloid cells ( CD68 ) and plasma cells ( JCHAIN ) .

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Figure 1c, Log normalised expression of markers for epithelial (EPCAM), mature luminal epithelial (ESR1), myoepithelial (KRT5, KRT14 and ACTA2) and proliferating cancer cells (MKI67).e, Log normalised expression of markers for fibroblasts (PDGFRB, THY1, COL1A1, ITGB1 and S100A4), endothelial cells (PECAM1), T-cells (CD3D), CD8 T cells (CD8A), T-regulatory cells (FOXP3), B-cells (MS4A1), myeloid cells (CD68) and plasma cells (JCHAIN).


Figure 2c , Log expression of parenchymal gene markers commonly associated with CAFs and perivascular cells .

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Figure 2c, Log expression of parenchymal gene markers commonly associated with CAFs and perivascular cells.


Figure 3b , Candidate transcriptional drivers of each CAF and PVL subset . Violin plots showing the log normalised gene expression ( left ) of the TF and its respective AUC regulon activity ( right ) . TFs ZEB1 and FOXP1 enriched in myofibroblast - like CAFs , EGR2 and TCF7L2 enriched in inflammatory - like CAFs , MEF2C enriched in PVL cells and NR2F2 enriched in immature - PVL cells .

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Figure 3b, Candidate transcriptional drivers of each CAF and PVL subset. Violin plots showing the log normalised gene expression (left) of the TF and its respective AUC regulon activity (right). TFs ZEB1 and FOXP1 enriched in myofibroblast-like CAFs, EGR2 and TCF7L2 enriched in inflammatory-like CAFs, MEF2C enriched in PVL cells and NR2F2 enriched in immature-PVL cells.


Figure 4c - d , Immunofluorescence of cultured human CAFs ( c ) and PVL cells ( d ) from passage 8 , staining for CD34 ( CAFs ) , ⍺ - SMA ( myCAFs and PVL cells ) , CD146 ( PVL cells ) and CD36 ( imPVL cells ) . e - f , Quantitative analysis of collagen abundance ( e ) and orientation ( f ) using second harmonic generation ( SHG ) from cellular derived matrices from stromal subsets and representative images multiphoton SHG images ( n = 3 biological replicates ) . All SHG intensity values within each replicate were normalised to the SHG intensity of the myCAFs . Error bars represent standard deviation . Statistical significance for collagen abundance ( e ) was determined using unpaired two - tailed Student ' s t test with equal standard deviation with p ‐ values denoted by asterisks : * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 . After normalization of the orientation peak distributions ( f ) , statistical significant was determined using a Kruskal - Wallis test with Dunn ' s post - hoc multiple comparisons test ( p value < 0 . 0001 ) . , Immunohistochemical staining of PDGFRβ , ⍺ - SMA , CD34 and CD146 in serial sections cut 4 μm apart from matched cases ; Patient - 2 ( g ) Images were aligned using FIJI . Tumour ( T ) regions are annotated by the solid yellow line . Co - localisation of CD34 and CD146 was used to distinguish blood vessels ( BV ) , where their differential staining was used to identify CAFs and PVL cells . g , MyCAFs were found to be localised at the invasive stromal interface ( insert A ) , whilst iCAFs were located at distal regions ( insert B ) with a high abundance of tumour infiltrating lymphocytes ( TILs ) . Immunohistochemical staining of PDGFRβ , ⍺ - SMA , CD34 and CD146 in serial sections cut 4 μm apart from matched cases ; Patient - 4 ( h ) . Images were aligned using FIJI . Tumour ( T ) regions are annotated by the solid yellow line . Co - localisation of CD34 and CD146 was used to distinguish blood vessels ( BV ) , where their differential staining was used to identify CAFs and PVL cells . h , Case with a high abundance of perivascular - like ( PVL ) cells in regions surrounded by blood vessels .

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Figure 4c-d, Immunofluorescence of cultured human CAFs (c) and PVL cells (d) from passage 8, staining for CD34 (CAFs), ⍺-SMA (myCAFs and PVL cells), CD146 (PVL cells) and CD36 (imPVL cells).e-f, Quantitative analysis of collagen abundance (e) and orientation (f) using second harmonic generation (SHG) from cellular derived matrices from stromal subsets and representative images multiphoton SHG images (n = 3 biological replicates). All SHG intensity values within each replicate were normalised to the SHG intensity of the myCAFs. Error bars represent standard deviation. Statistical significance for collagen abundance (e) was determined using unpaired two-tailed Student's t test with equal standard deviation with p‐values denoted by asterisks: *p < 0.05, **p < 0.01 and ***p < 0.001. After normalization of the orientation peak distributions (f), statistical significant was determined using a Kruskal-Wallis test with Dunn's post-hoc multiple comparisons test (p value < 0.0001)., Immunohistochemical staining of PDGFRβ, ⍺-SMA, CD34 and CD146 in serial sections cut 4 μm apart from matched cases; Patient-2 (g) Images were aligned using FIJI. Tumour (T) regions are annotated by the solid yellow line. Co-localisation of CD34 and CD146 was used to distinguish blood vessels (BV), where their differential staining was used to identify CAFs and PVL cells. g, MyCAFs were found to be localised at the invasive stromal interface (insert A), whilst iCAFs were located at distal regions (insert B) with a high abundance of tumour infiltrating lymphocytes (TILs).Immunohistochemical staining of PDGFRβ, ⍺-SMA, CD34 and CD146 in serial sections cut 4 μm apart from matched cases; Patient-4 (h). Images were aligned using FIJI. Tumour (T) regions are annotated by the solid yellow line. Co-localisation of CD34 and CD146 was used to distinguish blood vessels (BV), where their differential staining was used to identify CAFs and PVL cells. h, Case with a high abundance of perivascular-like (PVL) cells in regions surrounded by blood vessels.


Figure 5c - e , Imputed gene expression of selected candidate signalling molecules identified between the four stromal subsets and malignant ( c ) epithelial , ( d ) myeloid and ( e ) T - cells . Expression of ligands in stromal clusters are represented on the left , with cognate receptors on target cells on the right .

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Figure 5c-e, Imputed gene expression of selected candidate signalling molecules identified between the four stromal subsets and malignant (c) epithelial, (d) myeloid and (e) T-cells. Expression of ligands in stromal clusters are represented on the left, with cognate receptors on target cells on the right.


Figure 6b , Representative cohort ( METABRIC ) showing the prognostic value of iCAF T - cell dysfunction signature in the context of CTLs for a total of 233 patients . Kaplan - Meier ' s present two groups of patients , ' low CTL ' ( blue line ) and ' high CTL ' ( red line ) , as estimated according to the average expression of CTL - specific genes and stratified as compared to the mean . Tumours with low iCAF T - cell dysfunction signatures ( top ) show patients with high CTL levels have a better survival outcome . In contrast , this survival benefit is lost in tumours with a high iCAF T - cell dysfunction signature ( bottom ) . P - values were defined from the Cox proportional hazard ( Cox - PH ) model . dPVL profiles and CTL exclusion consistent in our study . g - h , Accurate quantification of CTLs ( g ) and representative immunohistochemistry staining for CD8 on matched patient tumour sections ( h ) . n = 5 stromal 1 mm2 regions were counted per tumour . The central band , boxes and whiskers represent the median , lower / upper quartile and min / max CTL counts per 1mm2 , respectively . P3 is shown as an example of a low dPVL profile with high CTLs . In contrast , P4 has a high dPVL profile with low CTLs . Statistical significance was determined using pairwise comparison with Student ' s t test with p ‐ values denoted by asterisks : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 .

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Figure 6b, Representative cohort (METABRIC) showing the prognostic value of iCAF T-cell dysfunction signature in the context of CTLs for a total of 233 patients. Kaplan-Meier's present two groups of patients, 'low CTL' (blue line) and 'high CTL' (red line), as estimated according to the average expression of CTL-specific genes and stratified as compared to the mean. Tumours with low iCAF T-cell dysfunction signatures (top) show patients with high CTL levels have a better survival outcome. In contrast, this survival benefit is lost in tumours with a high iCAF T-cell dysfunction signature (bottom). P-values were defined from the Cox proportional hazard (Cox-PH) model.dPVL profiles and CTL exclusion consistent in our study. g-h, Accurate quantification of CTLs (g) and representative immunohistochemistry staining for CD8 on matched patient tumour sections (h). n = 5 stromal 1 mm2 regions were counted per tumour. The central band, boxes and whiskers represent the median, lower/upper quartile and min/max CTL counts per 1mm2, respectively. P3 is shown as an example of a low dPVL profile with high CTLs. In contrast, P4 has a high dPVL profile with low CTLs. Statistical significance was determined using pairwise comparison with Student's t test with p‐values denoted by asterisks: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.


Figure 1Mouse qPCR array analysis was performed with Colo357 and MiaPaca - 2 orthotopic tumor samples treated with saline ( control ) or 2G8 ( n = 3 / group ) to detect changes of mouse stromal gene expression after treatment . A heat map was generated with ratios of gene expression ( 2G8 vs . control ) . ELISA - based Quansys mouse chemokine assay was performed with Capan - 1 ( Cap ) , MiaPaCa - 2 ( Mia ) , Colo357 ( Colo ) , and C5LM2 ( C5 ) orthotopic tumor samples treated with saline ( control ) or 2G8 ( n = 3 / group ) to detect mouse chemokine changes after treatment . Change of mouse IL - 6 was shown . KIC were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . Tumors from were collected for mouse IL - 6 ELISA ( n = 6 / group ) . KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 ( control ) or 2G8 . The activation of SMAD2 ( P - Ser465 / 467 ) ( E and H - I ) , STAT3 ( P - Tyr705 ) ( F and J - K ) and expression of IL - 6R ( G ) was detected by immunohistochemistry ( n = 4 / group ) .

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Figure 1Mouse qPCR array analysis was performed with Colo357 and MiaPaca-2 orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect changes of mouse stromal gene expression after treatment. A heat map was generated with ratios of gene expression (2G8 vs. control).ELISA-based Quansys mouse chemokine assay was performed with Capan-1 (Cap), MiaPaCa-2 (Mia), Colo357 (Colo), and C5LM2 (C5) orthotopic tumor samples treated with saline (control) or 2G8 (n = 3/group) to detect mouse chemokine changes after treatment. Change of mouse IL-6 was shown.KIC were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. Tumors from were collected for mouse IL-6 ELISA (n = 6/group).KIC mice were treated for 4 weeks and KPC mice were treated for 55 days with Mac84 (control) or 2G8. The activation of SMAD2 (P-Ser465/467) (E and H-I), STAT3 (P-Tyr705) (F and J-K) and expression of IL-6R (G) was detected by immunohistochemistry (n = 4/group).


Figure 2Single - cell RNA sequencing was performed to profile cell populations in normal mouse pancreas ( n = 2 ) , early KIC ( 40 - day - old , n = 2 ) and late KIC ( 60 - day - old , n = 3 ) pancreata . Samples from the same stage were pooled . Violin plots of expression of Il6 , Tgfbr1 and Tgfbr2 in distinct cell populations is shown . Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC ( mPLRB8 , mPLRB9 ) , KPC ( KPC - M01 , KPC - M09 ) and KPfC ( BMFA3 , CT1BA5 ) mouse cancer cells , mouse macrophages ( RAW 264 . 7 ) , and mouse fibroblasts ( NIH 3T3 and pancreatic stellate cells ) . RAW 264 . 7 cells were induced into M1 ( 30 ng / ml LPS for 18 hours ) or M2 ( 20 ng / ml IL - 4 for 18 hours ) macrophages . Tubulin was used as a loading control . Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA ( n = 223 ) using R .

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Figure 2Single-cell RNA sequencing was performed to profile cell populations in normal mouse pancreas (n = 2), early KIC (40-day-old, n = 2) and late KIC (60-day-old, n = 3) pancreata. Samples from the same stage were pooled. Violin plots of expression of Il6, Tgfbr1 and Tgfbr2 in distinct cell populations is shown.Expression of TGFβR1 and TGFβR2 in cell lysates harvested from KIC (mPLRB8, mPLRB9), KPC (KPC-M01, KPC-M09) and KPfC (BMFA3, CT1BA5) mouse cancer cells, mouse macrophages (RAW 264.7), and mouse fibroblasts (NIH 3T3 and pancreatic stellate cells). RAW 264.7 cells were induced into M1 (30 ng/ml LPS for 18 hours) or M2 (20 ng/ml IL-4 for 18 hours) macrophages. Tubulin was used as a loading control.Pearson and Spearman correlation of the expression of IL6 and TGFBR2 in PDA patients from TCGA (n = 223) using R.


Figure 3Control , M1 ( LPS stimulated ) and M2 ( IL - 4 stimulated ) RAW 264 . 7 cells , as well as NIH 3T3 cells were treated with TGFβ ( 30 ng / ml ) , or TGFβ + 2G8 ( 100 ng / ml ) for 24 hours . Cell lysates were harvested and blotted for P - SMAD2 ( P - Ser465 / 467 ) , SMAD2 , and tubulin ( A ) . Control , M1 ( LPS stimulated ) and M2 ( IL - 4 stimulated ) RAW 264 . 7 cells , as well as NIH 3T3 cells were treated with TGFβ ( 30 ng / ml ) , or TGFβ + 2G8 ( 100 ng / ml ) for 24 hours . Conditioned media ( CM ) was collected mouse IL - 6 ELISA ( B - C ) . NIH 3T3 ( D ) , pancreatic stellate cells ( PSC ) ( E ) and human CAF cell lines CAF - PC1 and CAF - PC2 ( F ) were treated with TGFβ ( 30 ng / ml ) and / or IL - 1α ( 1 ng / ml ) for 24 hours . CM was collected for mouse or human IL - 6 ELISA . CAF - PC2 cells were treated with TGFβ ( 30 ng / ml ) for 24 hours with siRNA - mediated knockdown of JUNB , JUND , SMAD4 or RELA . CM was collected and subjected to human IL - 6 ELISA . CAF - PC2 cells were treated with IL - 1α ( 1 ng / ml ) ( H ) for 24 hours with siRNA - mediated knockdown of JUNB , JUND , SMAD4 or RELA . M was collected and subjected to human IL - 6 ELISA . KIC ( mPLRB9 ) , KPC ( KPC - M09 ) and KPfC ( BMFA3 ) cell lines were treated with normal DMEM ( CTRL ) , CM from NIH 3T3 ( CM ) , CM from TGFβ - treated NIH 3T3 ( TGFβ - CM ) , CM from TGFβ - treated NIH 3T3 + 2G8 ( TGFβ - CM + 2G8 ) normal DMEM + TGFβ ( TGFβ ) , CM from TGFβ - treated NIH 3T3 + IL - 6 neutralizing antibody ( TGFβ - CM + IL - 6 Ab ) . Cell lysates were harvested and blotted for P - STAT3 ( P - Tyr705 ) , STAT3 , P - SMAD2 ( P - Ser465 / 467 ) , SMAD2 , and tubulin ( J ) . 3D culture : cells were seeded on poly - HEMA - coated 96 - well plates and cultured for 4 days ( 5000 cancer cells for monoculture , 3000 cancer cells + 2000 NIH 3T3 for co - culture ) . IL - 6 neutralizing antibody ( 100 ng / ml ) . 3D culture : cells were seeded on poly - HEMA - coated 96 - well plates and cultured for 4 days ( 5000 cancer cells for monoculture , 3000 cancer cells + 2000 NIH 3T3 for co - culture ) . IL - 6 neutralizing antibody ( 100 ng / ml ) .

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Figure 3Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Cell lysates were harvested and blotted for P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (A).Control, M1 (LPS stimulated) and M2 (IL-4 stimulated) RAW 264.7 cells, as well as NIH 3T3 cells were treated with TGFβ (30 ng/ml), or TGFβ + 2G8 (100 ng/ml) for 24 hours. Conditioned media (CM) was collected mouse IL-6 ELISA (B-C).NIH 3T3 (D), pancreatic stellate cells (PSC) (E) and human CAF cell lines CAF-PC1 and CAF-PC2 (F) were treated with TGFβ (30 ng/ml) and/or IL-1α (1 ng/ml) for 24 hours. CM was collected for mouse or human IL-6 ELISA.CAF-PC2 cells were treated with TGFβ (30 ng/ml) for 24 hours with siRNA-mediated knockdown of JUNB, JUND, SMAD4 or RELA. CM was collected and subjected to human IL-6 ELISA.CAF-PC2 cells were treated with IL-1α (1 ng/ml) (H) for 24 hours with siRNA-mediated knockdown of JUNB, JUND, SMAD4 or RELA. M was collected and subjected to human IL-6 ELISA.KIC (mPLRB9), KPC (KPC-M09) and KPfC (BMFA3) cell lines were treated with normal DMEM (CTRL), CM from NIH 3T3 (CM), CM from TGFβ-treated NIH 3T3 (TGFβ-CM), CM from TGFβ-treated NIH 3T3 + 2G8 (TGFβ-CM + 2G8) normal DMEM + TGFβ (TGFβ), CM from TGFβ-treated NIH 3T3 + IL-6 neutralizing antibody (TGFβ-CM + IL-6 Ab). Cell lysates were harvested and blotted for P-STAT3 (P-Tyr705), STAT3, P-SMAD2 (P-Ser465/467), SMAD2, and tubulin (J).3D culture: cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). IL-6 neutralizing antibody (100 ng/ml).3D culture: cells were seeded on poly-HEMA-coated 96-well plates and cultured for 4 days (5000 cancer cells for monoculture, 3000 cancer cells + 2000 NIH 3T3 for co-culture). IL-6 neutralizing antibody (100 ng/ml).