Fig 2: DJ-1 promotes DSB repair. (A,B) Representative immunofluorescence images and quantification of γ-H2AX foci in DJ-1 KO and WT U2OS cells at the indicated times after ETO treatment. Cells were treated with ETO (10 µM, 1 h) and were allowed to recover in an ETO-free medium. Scale bar, 10 μm. Data are presented as the mean ± SEM, n > 150 cells. *, p < 0.05; **, p < 0.01; ns, nonsignificant (two-tailed t-test). (C,D) Representative immunofluorescence images and the quantification of γ-H2AX foci in WT and the stable expression of DJ-1 U2OS cells at the indicated times after ETO treatment. Scale bar, 10 μm. Data are presented as the mean ± SEM, n > 150 cells. ***, p < 0.001; ****, p < 0.0001; ns, nonsignificant (two-tailed t-test). (E,F) Quantification of tail moments in DJ-1 KO (E) or stable expression of DJ-1 (F) U2OS cells treated with ETO (10 µM, 1 h) as determined by a neutral comet assay. Data are presented as the mean ± SEM, n > 100 cells. *, p < 0.05; **, p < 0.01; ns, nonsignificant (two-tailed t-test). (G) Schematics of HR and NHEJ reporter assays. (H,I) Quantification of DJ-1 knockdown and overexpression on HR (H) and NHEJ (I) in HEK293T cells. HEK293T cells transfected with the indicated siRNA or plasmids were subjected to DR-GFP assay or EJ5-GFP assay analyzed by flow cytometry. Data are presented as the mean ± SEM, n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (two-tailed t-test). (J,K) Representative immunofluorescence images and quantification of RAD51 foci in DJ-1 KO and WT U2OS cells treated with ETO (10 µM, 1 h) at the indicated time points. Scale bar, 10 μm. Data are presented as the mean ± SEM, n > 150 cells. *, p < 0.05; ****, p < 0.0001; ns, nonsignificant (two-tailed t-test). (L,M) Representative immunofluorescence images and quantification of 53BP1 foci in DJ-1 KO and WT U2OS cells treated with ETO (10 µM, 1 h) at the indicated time points. Scale bar, 10 μm. Data are presented as the mean ± SEM, n > 150 cells. **, p < 0.01; ns, nonsignificant (two-tailed t-test).

Fig 3: Cd ions specifically impeded the NHEJ repair pathway. (A) GC-2 cells after transient cotransfection with the NHEJ reporter and/or I-SceI treated with or without CdCl2 (20 μM). The percentage of GFP and RFP positive cells was analyzed by flow cytometry. n > 10000 cells for each group. (B) GC-2 cells after transient cotransfection with the HR reporter and/or I-SceI with or without CdCl2 (20 μM). The ratio of GFP+ to RFP+ cells, indicating the efficiency of HR repair, was analyzed by flow cytometry. n > 10000 cells for each group. (C) GC-2 cells were transfected with linearized-pCDH plasmids with or without CdCl2 (20 μM). The percentage of GFP-positive cells was analyzed by flow cytometry. n> 10000 cells for each group. (D) GC-2 cells were treated by H2O2 (40 μM) with or without Cd (20 μM) treatment for 2 h, followed by immunoblot analysis of 53BP1 and Rad51 on the chromatin fraction. (E) Representative images of immunofluorescence of 53BP1 in GC-2 cells were synchronized in G1 phase and Rad51 in S phase, were treated by H2O2 (40 μM) with or without Cd (20 μM) treatment for 2 h, cells were co-stained with DNA damage marker γ-H2AX. Scale bar, 5 μm. (F) Quantification of 53BP1 foci per cell treated as in (E). n > 200 cells for each group. (G) Quantification of RAD51 foci per cell treated as in E. n > 200 cells for each group. **P< 0.01; ***P < 0.001; ****P < 0.0001; ns indicates non-significant difference. Data presented as mean ± SD.

Fig 4: LAP induced cell senescence in HER2+ breast cancer cells. (A, B) Cell senescence development and morphology alteration in SK‐S cells, compared to parental SK cells. SK cells were treated with LAP at 250 nM for 7 days and subjected to SA‐β‐Gal staining. (C, D) Cell cycle distribution of SK and SK‐S cells determined by flow cytometry. (E) Expression of DNA damage foci in SK and SK‐S cells detected by immunofluorescence. γH2AX (green), 53BP1 (red), and nuclear DNA (blue) counterstained with DAPI (scale bar: 10 μm). Magnification: ×630. (F, G) Gene expression of senescence markers in SK and SK‐S cells measured by Real‐time PCR. SK, parental SKBR3 cells; SK‐S, senescent SKBR3 cells. *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001. Data are expressed as mean ± SD (n = 3).

Fig 5: Activation of HR is limited and delayed in MM134 (ILC) versus MCF7 (IDC) cells.(A) MM134 cells treated with 10μM etoposide for 4 or 24 hours were analyzed by western blot for canonical interactors of MDC1 (yH2AX, ATM, and MRN) and down-stream mediators of canonical MDC1 signaling (CHK1/2, 53BP1, RAD51, and DNAPKcs). (B) Western blot comparison of MM134 (ILC) and MCF7 (IDC) for MDC1 interaction of downstream NHEJ (53BP1 and DNAPKcs) vs HR (Rad51) was performed. Increased turnover of RAD51 in MCF7 is suggestive of HR being used to repair etoposide induced double strand breaks. (C) Breast cancer cells were treated with a 4hr pulse of 10μM etoposide, or 4Gy ionizing radiation, and allowed to recover for 4 or 24hr prior to assessing RAD51 foci formation by immunofluorescence. Points represent foci counts in individual nuclei, read line represent median per condition. (D) Immunoprecipitation of MDC1 in MM134 and MCF7 cells treated with 10μM etoposide. Increased co-IP of RAD51 is observed in MCF7 but not MM134. (E) TrafficLight DNA repair reporter output from breast cancer cells. Bars represent mean ± SD form n=5 biological replicates. Red bar represents ANOVA p<0.05 for indicated comparison.