Fig 2: miR-134 enhances DNA repair efficiency by promoting H2AX S139 phosphorylation. (a) Left, representative comet assay revealing the formation of DNA breaks (formation of a ‘comet tail') in miR-134-transfected T29 and SKOV3 cells treated with adriamycin (0.5 µM) for 24 h and then allowed to recover for 12 h. Right, box plot graph showing Tail DNA%. (b) ChIP assay with anti-?-H2AX was performed 24 h after SKOV3-Hygro-EGFP cells were co-transfected with miR-134 or the control mimics. The genomic locations of the qPCR primers are referred to as ChIP primers in Supplementary Figure S6a. (c) The proportion of EGFP-positive cells was determined 48 h after SKOV3-Hygro-EGFP cells were co-transfected with the pCBASceI plasmid and miR-134 or the control mimics. (d) Representative western blot images of ?-H2AX and H2AX in H2AX-S139A-expressing cells or control cells 24 h after adriamycin (0.5 µM) treatment. (e) Representative western blot images of ?-H2AX and H2AX in cells co-transfected with the H2AX-S139A plasmid and miR-134 or the control mimics for 48 h. (f) The box plot graph showing tail DNA% in H2AX-S139A-transfected cells treated with adriamycin (0.5 µM) for 24 h and then allowed to recover for 12 h. (g) ChIP assay using anti-?-H2AX was performed 24 h after SKOV3-Hygro-EGFP cells were co-transfected with pCBASceI and the H2AX-S139A plasmid or the control plasmid. (h) The proportion of EGFP-positive cells was determined 48 h after SKOV3-Hygro-EGFP cells were co-transfected with the pCBASceI plasmid and the H2AX-S139A plasmid or the control plasmid. (i) The box plot graph depicting tail DNA% in T29 cells co-transfected with miR-con/miR-134 and pcDNA3.1/H2AX-S139A after a 24 h adriamycin treatment and a 12-h recovery. (j) The proportion of EGFP-positive cells was determined 48 h after SKOV3-Hygro-EGFP cells were co-transfected with miR-con/miR-134 and pcDNA3.1/H2AX-S139A. Data are shown as mean±S.D. from three independent experiments. *P<0.05, **P<0.01 by Student's t-test

Fig 3: BRCA1-I15 promotes PARPi resistance.a BRCA1 and RAD51 foci formation was assessed by immunofluorescence (IF) with or without 10 Gy IR. See Supplementary Fig. 3b for representative images as well as ?-H2AX and 53BP1 controls. b HR repair capacity assessed by DR-GFP assay. **p < 0.01 compared to 293T cells. See Supplementary Fig. 3c-d for representative histograms and summary. c MDA-MB-231 and SNU-251-RR cells were treated with scrambled (Sc), BRCA1 exon 11 (E11) or intron 15 (I15)-targeting siRNA and BRCA1 protein expression assessed by Western blotting. d Cells were treated as in panel (c) and examined by IF for BRCA1 and RAD51 foci post 10 Gy IR. ***p < 0.001 compared to Sc-treated cells. See Supplementary Fig. 3e for representative images. e Cells were treated as in panel (c) and subject to increasing concentrations of rucaparib and colony formation assessed. Data are the mean ± SD of n = 3 biological replicates. Statistical significance was assessed by unpaired, two-tailed t tests.

Fig 4: Meflufen induces DNA damage and apoptosis in cancer cells. (A) D492, D492HER2, and MDA-MB231 cells were seeded on a six well plate and incubated with either none, 1 µmol L-1 or 5 µmol L-1 melflufen for 30 min and harvested 3 h post-treatment. Equally loaded protein lysates subjected for immunoblotting for H2AX/ yH2Ax One representative blot of three is shown. (B) Quantification of protein bands of three independent experiments performed in 4A. Actin was used as loading control. Quantification was perfomed by FIJI. Shown are means ± standard deviation (n = 3). (C) D492, D492HER2, and MDA-MB231 cells were incubated with 1 µmol L-1 of melflufen for 30 min and harvested on indicated timepoints and subjected for immunoblotting for H2AX/ yH2Ax. One representative blot of 3 is shown. (D) Quantification of protein bands of three independent experiments performed in 4C. Actin was used as loading control. Quantification was perfomed by FIJI. Shown are means ± standard deviation (n = 3). (E) D492, D492HER2, and MDA-MB231 cells were cultured in monolayer and 24 h after treatment with none, 1 µmol L-1 or 5 µmol L-1 melflufen or melphalan, cells were fixed and stained for yH2Ax (red). IF analysis reveals that melflufen increases DNA damage in a dose dependent manner. Melphalan shows only a minor increase in staining at the concentrations used. Scalebar = 30 µm. (F) For immunostaining of D3 cultures, colonies of D492, D492HER2 and MDA-MB231 were incubated with either 10 µmol L-1 melflfufen or melphalan for 2h, fixed 72h post-treatment and stained for yH2Ax (red) and DAPI (blue). Scalebar = 100 µm (G) D492HER2 and MDA-MB231 cells were incubated with increasing amounts of melflufen for 24 h, harvested in RIPA buffer and subjected to immunoblotting for cleaved caspase-3. One representative blot of 3 is shown. (H) Quantification of protein bands of 3 independent experiments performed in 4G for caspase-3. Actin was used as loading control. Quantification was perfomed by FIJI. Shown are means ± standard deviation (n = 3)

Fig 5: RhoA LoF differentially affects the phosphorylation of H2AX, Chk1, and p53 throughout the DDR signaling in NER- and TLS-deficient cells after UV-radiation stress. Immunoblottings showing the levels of expression and phosphorylation kinetics for Chk1, H2AX, and p53 in MRC5 (A), XPA-deficient (B), XPC-deficient (C), and XPV-deficient (D) cells up to 6 h after the irradiation with 6 J/m2 UVC, without or with previous RhoA inhibition by C3 toxin. The blots quantification is numerically shown under each band. Blots are representative of three independent experiments.