Fig 2: DJ-1 stimulates PARP1 activity. (A) Representative western blot and quantification of PAR and PARP1 proteins in DJ-1 KO and WT U2OS cells. Data are presented as the mean ± SD, n = 3. *, p < 0.05 (two-tailed t-test). (B) In vitro PARP1 enzymatic activity assay in the presence of recombinant DJ-1 protein. Data are presented as the mean ± SEM, n = 3. ***, p < 0.001; ****, p < 0.0001 (two-tailed t-test). (C) Representative western blot and quantification of PAR in WT and DJ-1 KO U2OS cells treated with ETO (10 µM) at the indicated time points. Data are presented as the mean ± SD, n = 3. **, p < 0.01; ***, p < 0.001 (two-tailed t-test). (D) Representative western blot and quantification of PAR in U2OS cells expressing Flag-DJ-1 following ETO (10 µM) at the indicated time points. Data are presented as the mean ± SD, n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, nonsignificant (two-tailed t-test). (E) Quantification of NHEJ and HR in HEK293T cells expressing Flag-DJ-1 in the presence and absence of PJ34 (10 µM). 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). (F) Representative immunofluorescence images and quantification of γ-H2AX in WT and stable expression of DJ-1 U2OS cells. U2OS were treated with ETO (10 µM, 1 h) in the presence and absence of PJ34 (10 µM) and were allowed to recover for 8 h. Scale bar, 10 μm. Data are presented as the mean ± SEM, n > 150 cells. *, p < 0.05; **, p < 0.01 (two-tailed t-test).

Fig 3: 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 4: DJ-1 responds to DNA damage and localizes to DSB sites. (A) Representative western blot and quantification of DJ-1 in U2OS cells treated with ETO (10 µM), CPT (10 µM), and MMS (0.01%) at the indicated timepoints. n = 3. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (two-tailed t-test). (B) Transcriptional analysis of DJ-1 in U2OS cells treated with ETO (10 µM) at the indicated timepoints by qPCR. GAPDH was used to normalize expression. Data are presented as the mean ± SEM, n = 3. ***, p < 0.001 (two-tailed t-test). (C) Representative immunofluorescence images of DJ-1 in U2OS cells exposed to ETO (10 µM) for 1 h. Scale bar, 10 µm. n = 3. (D) Representative western blot and quantification of endogenous DJ-1 in the cytoplasmic and nuclear fractions of U2OS cells treated with ETO (10 µM) at the indicated times. n = 3. *, p < 0.05; **, p < 0.01; ns, nonsignificant (two-tailed t-test). (E) Representative quantification of DJ-1 foci as in C. Data are presented as the mean ± SEM, n > 50 cells. ****, p < 0.0001 (two-tailed t-test). (F) Representative immunostaining of γ-H2AX and DJ-1 following ETO (10 µM, 30 min) treatment in U2OS cells. Scale bar, 10 μm. n = 3. (G) Representative PLA signals between γ-H2AX and DJ-1 in U2OS cells following ETO (10 µM, 30 min) treatment. Quantification of average PLA signals is shown at right. Scale bar, 10 μm. Data are presented as the mean ± SEM, n > 50 cells. ***, p < 0.001 (two-tailed t-test).

Fig 5: DJ-1 deficiency leads to DNA damage accumulation and defective PARP1 activity in PD fibroblasts. (A) Representative immunofluorescence images of DJ-1 in WT and DJ-1 mutant fibroblasts. Scale bar, 10 μm. (B) Representative western blot and quantification of γ-H2AX in WT and DJ-1 mutant fibroblasts. Data are presented as the mean ± SD, n = 3. *, p < 0.05 (two-tailed t-test). (C) Representative immunofluorescence images and quantification of γ-H2AX foci in WT and DJ-1 mutant fibroblasts 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). (D) Representative PLA signals between DJ-1 and PARP1 in fibroblasts following ETO (10 µM, 1 h) treatment. Quantification of average PLA signals are shown on the right. Scale bar, 10μm. Data are presented as the mean ± SEM, n > 100 cells. **, p < 0.01 (two-tailed t-test). (E) Representative western blot and quantification of PAR in WT and DJ-1 mutant fibroblasts treated with ETO (10 µM) at the indicated time points. Data are presented as the mean ± SD, n = 3. **, p < 0.01 (two-tailed t-test).