Fig 2: Ceritinib synergizes with PARPis in HR-proficient and -deficient ovarian cancer cells. A–E, OVCAR-8 (A–C) or COV362 (D and E) cells were plated as single cells and cultured in the continued presence of indicated concentrations of ceritinib and olaparib (A, B, D, and E) or veliparib (C) for 8–10 days to form colonies. Colonies were stained with Coomassie Blue and those with >50 cells/colony were counted manually. Graphs (A, C, and D) are representative of three independent experiments. Error bars, means ± SD of three technical replicates in the representative experiment. Combination index (CI) values (B and E) were calculated using the Chou–Talalay method in CalcuSyn software. CI > 1, CI = 1, and CI < 1 are antagonistic, additive, and synergistic, respectively. CI values (B and E) show the mean ± SEM for the three independent experiments. F, OVCAR-8 cells were transfected with control luciferase (siLUC) or a RAD51C siRNA pool. Forty-eight hours later, the cells were harvested, immunoblotted for RAD51C and HSP90 (loading control), and subjected to colony formation assays as in A, C, and D. Graph is representative of three independent experiments. Error bars, means ± SD of three technical replicates in the representative experiment.

Fig 3: Functional characterisation of RAD51C p.Arg312Trp missense variant. (A) RT–PCR expression analysis of RAD51C transgenic cDNA in RAD51C-deficient CL-V4B cells transduced with wild-type (CL-V4B RAD51C WT) or p.Arg312Trp–containing (CL-V4B RAD51C.p.Arg312Trp) human RAD51C. CL-V4B (CL-V4B EV) and RAD51C wild-type expressing V79B cells (V79B EV) transduced with empty vector were included as controls. Primers were designed to specifically amplify human RAD51C. (B) Verification and quantification of RAD51C protein expression levels in transgenic cells by using immunoblotting analysis with an anti-RAD51C antibody that recognises human and hamster RAD51C. CL-V4B RAD51C WT, CL-V4B RAD51C.p.Arg312Trp and control CL-V4B EV and V79B EV cells were treated with increasing MMC (C) and Olaparib (E) doses and survival was examined after 5 days of treatment. Values represent means±standard error of the mean (s.e.m.) of four independent experiments. Representative visual examination of cell survival assays by using crystal violet staining of MMC-treated (D), Olaparib-treated (F) and vehicle-treated cells. Plating efficiency differences between CL-V4B and V79B were accounted for by normalising each treated V79B and CL-V4B cell counting with its corresponding untreated (control) point. Cell cycle analysis of CL-V4B RAD51C WT, CL-V4B RAD51C.p.Arg312Trp and control cells treated with MMC (50 nM) (H) or vehicle (–) (G). Low panels show percentage of cells in G1, S and G2/M phases in each cell line. Data are means of four independent experiments. A full colour version of this figure is available at the British Journal of Cancer journal online.

Fig 4: Spanish non-BRCA1/2 high-risk breast and ovarian cancer family selected for Whole Exome Sequencing (WES) study. (A) Proband is highlighted by a black arrow. Individuals with tumours and ages of diagnosis are shown. (B) Electropherograms of RAD51C sequence spanning the c.934C>T variant show the presence of the c.934C>T variant in the proband’s blood sample. Sequence traces confirms loss of heterozygosity (LOH) in the proband’s (C) and her sister’s (D) ovarian tumours and RAD51C wild-type sequence in normal tissue from the proband’s father (E). A full colour version of this figure is available at the British Journal of Cancer journal online.

Fig 5: OLA induces DNA damage(A) Sertoli cells were treated with the indicated concentrations of OLA for 24 h, and treated with vehicle (0.2% DMSO) were used as negative control followed by immunblot analysis for the expression of ?H2A.X and Rad51C. Semiquantitative analyses of protein expression in following histogram (mean ± S.E.M., three independent replicates per groups). * p < 0.05; ** p < 0.01. (B) Representative images showing the immunofluorescence staining of ?H2A.X (red) and DAPI (nuclei, blue) in Sertoli cells after treatment with the indicated concentrations of OLA for 24 h. Scale bar, 20µm. (C) Sertoli cells were pretreated with NAC (10 mM) for 1 h, followed by 400 µg/ml OLA treatment for 24 h prior to immunblot analysis for the expression of ?H2A.X. GAPDH served as protein loading control. Semiquantitative analysis of protein expression in following number (mean ± S.E.M., three independent replicates per groups). * p < 0.05; ** p < 0.01.