Fig 1: Validation cohort tissue-resolution explant DDR capacities with associated cytotoxicity and metabolic classifications.a DNA damage response (DDR) pathway states for Validation cohort explants. Each column is an explant and each row is a DDR pathway capacity. A range of defective pathway signatures are evident. Clustering resolves Host-Cell Reactivation (NHEJ) and Rad51 (HR) assays to almost diametrically opposed DSB repair capacity states. Aggregate pathway competences sum double-strand break, single-strand break, and all strand break capacities. Intra-explant class-balance bar plots depict extent-of-cell-viability survivorship following repair blockade, and during genomic assault. b Explant platinum cytotoxicity was completely classified by the five DDR pathway capacity states (discriminant analysis ROC AUC of 1). c The five DDR pathway capacity states completely classify mitochondrial membrane health (discriminant analysis ROC AUC of 1). d Explant response to ROS assaults was described by the five DDR pathway capacity states (AUC of 1) with distinct but limited discrimination distances (left plot). A marked increased discriminatory distance was obtained when supplemented with mitochondrial membrane status (right plot). An explant mitochondrial membrane state and its ability to recover a ROS burden were inversely correlated (P = 0.0390).
Fig 2: The TMZ/Nira regimen suppresses plasmacytoma xenograft in vivo. (a) Human MM cell-derived xenograft model was built using RPMI8226 cells to confirm the synergistic effects of TMZ and Nira. The animals had decreased tumor volumes upon administration of the TMZ-Nira combination compared with the TMZ and Nira monotherapy and control groups. (b, c) Tumor size and weight measurements revealed decreased plasmacytoma xenograft growth after TMZ/Nira cotreatment. (d) The percentages of TUNEL-positive cells were obtained by ImageJ. (e) Apoptotic and proliferating cells in tumor specimens, respectively, were detected by the TUNEL assay, H&E staining, and immunochemistry. The expression levels of ?H2A.X, RAD51, Ki67, and cleaved caspase-3 were determined by immunohistochemistry. Reduced expression of Ki67 and elevated expression of ?H2A.X, RAD51, and cleaved caspase-3 were found in the TMZ plus Nira group. Mean ± SD (n = 5 per group). Significant differences were indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus control group; ###P < 0.001 versus TMZ group; +++P < 0.001 versus Nira group. Scale bar, 20 µm. Original magnification, ×400. TMZ: temozolomide; Nira: niraparib.
Fig 3: Overexpression of RAD51 abolishes CTBP1 knockdown-induced effects on proliferation and apoptosis of DDP-resistant gastric cancer cells. (A) Western blot analysis was performed to detect RAD51 protein expression levels in AGS/DDP cells following transfection with RAD51 plasmid. *P<0.05 vs. pcDNA3.1. (B) The protein expression levels of CTBP1 and RAD51 following transfection with siCTBP1-2 with or without pc-RAD51 co-transfection was determined using western blot analysis. (C) Viability of AGS/DDP cells were evaluated using the Cell Counting Kit-8 assay. (D) Colony formation assay was performed to detect the proliferation of AGS/DDP cells. (E) Flow cytometric analysis was performed to detect the apoptosis of AGS/DDP cells. (F) Western blot analysis was performed to detect the protein expression levels of apoptosis-related proteins in AGS/DDP cells. ***P<0.001 vs. siNC; ##P<0.01, ###P<0.001 vs. siCTBP1-2 + pcDNA3.1. CTBP1, C-terminal-binding protein 1; DDP, cisplatin; RAD51, DNA repair protein RAD51 homolog 1; si, small interfering RNA.
Fig 4: RAD51 knockdown results in mitochondrial dysfunction. (A) MitoSOX distribution measured by flow cytometry in A2780, HO8910 and HEY cells transfected siScr or siRAD51 for 48 h. (B) ROS distribution measured by flow cytometry in A2780, HO8910 and HEY cells transfected siScr or siRAD51 treated with or without 500 nM MitoQ. (C) Measurements of MMP (by JC-1) in A2780, HO8910 and HEY cells transfected siScr or siRAD51 for 48 h. (D) Oxygen consumption rate (OCR) of A2780 cells transfected siScr or siRAD51 for 48 h. The OCR was measured under baseline conditions and after oligomycin, FCCP and rotenone/antimycin injection, as indicated by the arrows. (E) Extracellular acidification rate (ECAR) of A2780 cells transfected siScr or siRAD51 for 48 h. The ECAR was measured under baseline conditions and after glucose, FCCP and 2-deoxy-d-glucose (2-DG) injection, as indicated by the arrows. Data presented as mean ± S.D. are representative of three independent experiments. The statistical differences between the two groups were analyzed by two-sided unpaired Student's t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ns stand for no significant).
Fig 5: BARD1 ANK and BRCT domains promote BRCA1 foci formation.a MDA-MB-436 cells expressing BFP, BRCA1-full-length (FL), BRCA1-?RING (?R), BRCA1-C61G (C61), BRCA1-I26A+L63 A+K65A (3A), BRCA1-?RING-L- BARD1-?RING (?R?R) were assessed for BRCA1 and BARD1 expression by western blotting. b Cells from (a) were assessed for BRCA1 foci 8 h post IR by immunofluorescence. Above; representative images (scale bar, 10 µm). Left; mean and S.E.M. percentage foci positive cells; middle, foci positive cells were assessed for the number of foci present in a single nucleus, red line: median value; right, foci positive cells were assessed for the mean size of foci present per nuclei, n = 3 biological replicates. Foci positive cells were determined to be those with =10 foci/nuclei. ***p < 0.001, **p < 0.01, *p < 0.05, ns not significant (percentage positive cells: unpaired, two-tailed t-tests size; number and size per nucleus: nonparametric Mann–Whitney tests). See Source data for additional information. c Cells from (a) were assessed for RAD51 foci 8 h post IR by immunofluorescence. See Supplementary Fig. 3c for representative images. Mean and S.E M. percentage (=5) foci positive cells, n = 3 biological replicates. ***p < 0.001, **p < 0.01, ns not significant compared to FL (unpaired, two-tailed t-tests). d Cells from (a) were incubated with increasing concentrations of rucaparib for 2 weeks and colony formation assessed. Mean and S.E.M. colony formation as well as mean IC50 values, n = 4 biological replicates. e MCF7 BARD1-/- cells expressing GFP control, GFP-BARD1-full-length (FL), GFP-BARD1-?RING (?R), GFP-BARD1-?ANK (?A), GFP-BARD1-?BRCT (?B), and GFP-BARD1-R99E (R99E) were assessed for GFP and BRCA1 expression by western blotting. f Cells from (e) were assessed for BARD1 and BRCA1 foci 8 h post IR by immunofluorescence. Left; representative foci (scale bar, 10 µm). Right; foci analyses as described in (b). g Cells from (e) were assessed for RAD51 foci as described in (c). See Supplementary Fig. 3h for representative images. h Cells from (e) were incubated with increasing concentrations of rucaparib for 2 weeks and colony formation assessed. Mean and S.E.M. colony formation as well as mean IC50 values, n = 3 biological replicates.
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