Fig 2: GRB2 recruits MRE11 to the DNA damage sites.(A) GFP-MRE11 was transfected into the indicated stable cells, followed by UV-LMI imaging. Scale bars, 10 µm. (B) GFP-MRE11 track intensity values from 20 cells at every 30 s were quantified with ImageJ, and normalized average intensities are shown with the SD. (C) Kinetics of GFP-RPA70 recruitment to micro-irradiated regions in HeLa cells. GFP-RPA70 was transfected into the indicated stable cells, followed by UV-LMI imaging. Track intensities were calculated as described in (B). (D) HeLa cells exposed to 5-Gy IR (152 s) were either immediately lysed (0 min) or allowed to recover for the indicated time. The chromatin fractions and the soluble nuclear fractions were analyzed with indicated antibodies. (E) Cells were either untreated (UT) or irradiated with 5-Gy IR (152 s) and then lysed immediately (0 min) or allowed to recover for the indicated time. Chromatin fractions were analyzed by Western blot with the indicated antibodies (left). Comparison of GRB2-KO HeLa cells reconstituted with either WT (KO + GRB2) or K109R (KO + K109R) mutant (middle). Comparison of GRB2-KO HeLa cells reconstituted with either WT (KO + GRB2) or K109A (KO + K109A) mutant (right).

Fig 3: GRB2 KO sensitizes cells to PARPi.(A) MRE11 depletion levels with two different shRNA (KD1 and KD2) in U2OS cells with normal level of GRB2 (Ctrl) and GRB2-KO. (B) DR-GFP reporter assay in MRE11-KD control (Ctrl) and GRB2-KO U2OS cells. (C) DR-GFP reporter assay in PTEN-KD and/or GRB2-KO U2OS cells. (D) Sulforhodamine B assay of olaparib-induced cytotoxicity in control HeLa cells, GRB2-KO cells, and GRB2-KO cells reconstituted with WT or K109R mutant GRB2. IC50 values are shown. (E) Clonogenic assay evaluating the effect of a PARPi on colony formation of HeLa cells. Cells were treated with the indicated concentrations of the PARPi olaparib and cultured for 10 days, and then the resulting colonies were visualized with crystal violet staining and counted by GelCount instrument. Parental control (WT), GRB2-KO (KO), WTGRB2 (KO + GRB2), and K109RGRB2 (KO + K109R) reconstituted HeLa cells. (F) Bar graph representing annexin V/propidium iodide (PI) combined labeling and flow cytometry analysis of olaparib-treated control (WT), GRB2-KO, reconstituted WTGRB2 (KO + GRB2), and K109R mutant (KO + K109R) HeLa cells. Cells were treated with either dimethyl sulfoxide (DMSO) or 50 µM olaparib for 72 hours. All data are representative of three independent experiments. The significance was analyzed by two-sided Student’s t test. *P = 0.05, **P = 0.01, and ***P = 0.001; NS, not significant.

Fig 4: Effects of resection factors on foci pairing.(a) Frequency of paired 53BP1 foci in MRE11-depleted cells (G1 phase). BJ-hTERT cells were transfected with the indicated siRNA, and after 3 days the cells were irradiated with 2 Gy ionizing radiation and then fixed at the indicated time points. Plotted are the percentages of paired foci among the total foci in individual cells. Statistical analyses were performed using the two-tailed Mann-Whitney test (alpha = 0.05). Red bars represent means. (b) Confirmation of MRE11 depletion by western blotting (cropped blot). (c) Frequency of paired 53BP1 foci in CtIP-depleted cells (G1 phase). Transfection of siRNA, sample preparation, examination of foci pairing, and statistical analyses were performed as described for the MRE11-depleted cells in Fig. 4a. (d) Confirmation of CtIP depletion by western blotting (cropped blot). Full-length blots for Fig. 4b and d are shown in Supplementary Figure S7a,b and S7c,d, respectively.

Fig 5: YARS promoted homologous recombination and insensitized responses to PARP inhibitors in gastric cancer. For HGC-27/AGS cells, changes of the homologous recombination pathway representative components (ATM, BRCA1, MRE11, 53BP1, RAD51) after a YARS depletion or b YARS overexpression were assessed by western blot. After YARS depletion in GC cells, 48 h responses to the concentration cascades of c three chemotherapy agents (cisplatin, 5-FU, paclitaxel) or d two PARP inhibitors (Olaparib, Niraparib) were assessed with CCK-8 assay. v vector, Y YARS. *p < 0.05. ns not significant