Journal: Cell & bioscience

Article Title: MRE11:p.K464R mutation mediates olaparib resistance by enhancing DNA damage repair in HGSOC.

doi: 10.1186/s13578-023-01117-0

Figure Lengend Snippet: Fig. 3 MRE11:p.K464R mutation leads to Olaparib resistance in ovarian cancer cells. (A-B) The stable cell lines of SKOV3 (A) and A2780-MRE11WT/MRE11K464R (B) were constructed expressing MRE11WT and MRE11K464R protein and detected the MRE11 protein expression with West ern blot. (C-D) SKOV3 (C) and A2780 (D) MRE11WT /MRE11K464R cells were treated for 96 h with indicated dose of Olaparib and viability assessed by CCK8. (E-F) Representative pictures of colony formation assay in SKOV3 (E) and A2780 (F) MRE11WT /MRE11K464R cells treated with or without Olaparib for 10 days are in left. Relative colony formation rates of cell are presented as percent relative to DMSO (right). (G-H) SKOV3 (G) and A2780 (H) MRE11WT/MRE11K464R cells were treated with or without Olaparib for 48 h and then stained for γH2AX, MRE11, and DAPI (left), and quantified the γH2AX foci per cell (right). Scale bar, 25 μm. (I-J) SKOV3 (I) and A2780 (J) MRE11WT/ MRE11K464R cells were treated with or without Olaparib for 48 h and subjected to Comet analysis. DNA damage is quantified as percent DNA in tails. Each group represents at least 100 cells counted. Data are presented as mean values ± SEM from three independent experiments. ***p < 0.001, ****p < 0.0001, ns, not significant, as determined by the unpaired two-tailed Student’s t-test

Article Snippet: Human ovarian serous adenocarcinoma cell lines SKOV3 (ATCC® HTB-77TM) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Mutagenesis, Stable Transfection, Construct, Expressing, Colony Assay, Staining, Two Tailed Test

Journal: Cell & bioscience

Article Title: MRE11:p.K464R mutation mediates olaparib resistance by enhancing DNA damage repair in HGSOC.

doi: 10.1186/s13578-023-01117-0

Figure Lengend Snippet: Fig. 4 Interactions of MRE11:p.K464R with RAD50/RPS3. (A) Schematic diagram of IP-MS detection. (B) The directly-interacting proteins of MRE11 were screened in the String database according to the IP-MS data. (C-D) SKOV3- MRE11WT/MRE11K464R cells were treated with or without Olaparib for 24 h. The cells were then lysed and immunoprecipitation with MRE11 antibody (C) and the relative gray scale density of RAD50(up) and RPS3 (down) to MRE11 are presented in D.(E-F) SKOV3- MRE11WT/MRE11K464R cells were treated with Olaparib or DMSO. The cells were then lysed and immunoprecipitation with RPS3 antibody (E) and the relative gray scale density of RAD50 (up) and MRE11 (down) to MRE11 are presented in F. (G-H) Detection of MRE11-RAD50 (G) and MRE11-RPS3(H) interaction was carried out by PLA labeling in SKOV3 MRE11WT/MRE11K464R cells treated with or without Olaparib for 24 h. Rep resentative images are shown. Scale bars, 5 μm. The scatterplot displays quantification of the PLA signals per nucleus from at least 100 cells from three independent experiments. Data are mean ± SEM. (I-J) ER-AsiSI Hela cells were transfected with empty vector or MRE11WT or MRE11K464R, and then treated with 4-OHT to induce DSBs. RAD50 (I) and RPS3 (J) accumulation at DNA damage sites generated by AsiSI was detected by ChIP qPCR. Data are presented as mean values ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, as determined by the unpaired two-tailed Student’s t-test

Article Snippet: Human ovarian serous adenocarcinoma cell lines SKOV3 (ATCC® HTB-77TM) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Protein-Protein interactions, Immunoprecipitation, Labeling, Transfection, Plasmid Preparation, Generated, ChIP-qPCR, Two Tailed Test

Journal: Cell & bioscience

Article Title: MRE11:p.K464R mutation mediates olaparib resistance by enhancing DNA damage repair in HGSOC.

doi: 10.1186/s13578-023-01117-0

Figure Lengend Snippet: Fig. 5 Effects of MRE11_K464R mutation on NHEJ pathway. (A) Expression of NHEJ key proteins in SKOV3 MRE11WT /MRE11K464R cells after treated with Olaparib for 48 h, detected by Western Blot with indicated antibodies. (B) MRE11WT or MRE11K464R virus were infected into EJ5-Hela cells for 24 h. Then, the cells were transfected with an I-SceI expression plasmid for 48 h. NHEJ efficiency were determined by FACS. (C) ER-AsiSI Hela cells were infected with MRE11WT or MRE11K464R virus, and then treated with 4-OHT to induce DSBs. Ku70 accumulation at DNA damage sites generated by AsiSI was detect ed by ChIP qPCR. (D) A2780 MRE11WT /MRE11K464R cells after treated with Olaparib for 48 h. The chromatin fractions and the soluble nuclear fractions were analyzed with indicated antibodies by Western Blot. (E-F) A2780 MRE11WT /MRE11K464R cells were treated with or with Olaparib for 48 h, and co-stained with Ku70 (green) and γH2AX (red) antibodies. E The representative images of immunofluorescence are presented, F quantification of Ku70 and γH2AX co-localization ratio per cell. Each group represents at least 100 cells counted. Scale bar, 10 μm. (G) MRE11_K464R with or without siNC/siRAD50/siRPS3 were infected into EJ5-Hela cells for 24 h. Then, the cells were transfected with an I-SceI expression plasmid for 48 h. NHEJ efficiency were determined by FACS. (H) MRE11_K464R with or without siNC/siRAD50/siRPS3 were infected into ER-AsiSI Hela cells and then treated with 4-OHT to induce DSBs. Ku70 accumulation at DNA damage sites generated by AsiSI was detected by ChIP qPCR. (I) SKOV3 (left) and A2780 (right) MRE11K464R cells were treated for 96 h with indicated doses of Olaparib or SCR7 (up) / AZD7648 (down) alone or combined for 96 h and viability assessed. Data are presented as mean values ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, as determined by the unpaired two-tailed Student’s t-test

Article Snippet: Human ovarian serous adenocarcinoma cell lines SKOV3 (ATCC® HTB-77TM) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Mutagenesis, Expressing, Western Blot, Virus, Infection, Transfection, Plasmid Preparation, Generated, ChIP-qPCR, Staining, Immunofluorescence, Two Tailed Test

Journal: Cell & bioscience

Article Title: MRE11:p.K464R mutation mediates olaparib resistance by enhancing DNA damage repair in HGSOC.

doi: 10.1186/s13578-023-01117-0

Figure Lengend Snippet: Fig. 6 RPS3/RAD50 knockdown sensitizes K464R mutant cells to Olaparib. (A-B) SKOV3 (A) and A2780 (B) MRE11K464R cells were transfected with siRAD50 or siRPS3 alone or combine for 24 h and then treated for 96 h with indicated doses of Olaparib and viability was measured by CCK8. The expres sion of scramble siRNA (siNC) was used as control. (C-D) Cells were transfected with siRAD50 or siRPS3 alone or combine for 24 h and then treated with or without Olaparib for 10 days. The expression of siNC was used as control. Representative pictures of clonogenic assay in A2780 MRE11K464R cells (C). The mean clones of ROI are presented (D). (E-F) SKOV3 (E) and A2780 (F) MRE11K464R cells were transfected with siRAD50 or siRPS3 alone or combine for 24 h and then treated for 48 h and then subjected to Comet analysis. DNA damage is quantified as percent DNA in tails. The expression of siNC was used as control. Each group represents at least 150 cells counted. (G-H) SKOV3 (G) and A2780(H) MRE11K464R cells were transfected with siRAD50 or siRPS3 alone or combine for 24 h and then treated with or without Olaparib for 48 h and then stained for γH2AX (green) and DAPI (blue), and γH2AX foci-positive cells were quantified (below). Each group represents at least 150 cells counted. Scale bar, 20 μm. Data are presented as mean values ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, as determined by the unpaired two-tailed Student’s t-test

Article Snippet: Human ovarian serous adenocarcinoma cell lines SKOV3 (ATCC® HTB-77TM) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Knockdown, Mutagenesis, Transfection, Control, Expressing, Clonogenic Assay, Clone Assay, Staining, Two Tailed Test