Fig. 4

PRDX5 reverses SLUG-mediated BRCA2 silencing in dividing SLUG-positive BT549 cells. a Quantitative ChIP analysis comparing SLUG binding and PRDX5A binding to the BRCA2 silencer between dividing and quiescent cells. The data were normalized to respective IgG control antibodies and calculated as percent fold change over binding in quiescent cells (designated as 100%). The differences are statistically significant (p < 0.001). b ChIP assay showing the effect of H₂O₂ treatment on SLUG binding to the BRCA2 silencer. The cells were treated with the respective concentrations of H₂O₂ for 24 h prior to chromatin isolation and ChIP assay. M, 1 kb + DNA ladder; SL, pulldown with anti-SLUG antibody; IgG, pulldown with control anti-rabbit IgG; In, Input DNA before pulldown. c Dual luciferase reporter assay showing the effect of H₂O₂ treatment on the BRCA2 promoter-silencer activity in SLUG-positive BT549 and SLUG-negative MDA-MB-468 cells. The cells were transfected with the reporter construct and incubated for 16 h. Then, they were exposed to H₂O₂ for 24 h, after which the luciferase assay was performed. Untreated cells served as the control (100%) for each cell type. d Western blots showing the effect of H₂O₂ treatment on nuclear localization of PRDX5. CF, cytosolic fraction; NF, nuclear fraction. Fibrillarin, HSP90, and GSK3β served as a nuclear marker, a cytosolic marker, and the loading control, respectively. e Densitometry of western blots in D showing normalized levels of nuclear PRDX5. f Quantitative ChIP analysis showing the effect of increasing H₂O₂ concentrations on PRDX5 binding to the BRCA2 silencer. All values were normalized to those from respective IgG-controls and input-DNA. g Western blot showing siRNA-mediated knockdown of PRDX5. Control, scrambled_control_siRNA; KD, PRDX5_siRNA. β-actin served as a loading control. h Quantitative ChIP analysis of the effect of H₂O₂ treatment on SLUG binding to the BRCA2 silencer with or without PRDX5 knockdown. Results are mean ± SE (n = 3). The difference is statistically significant (p < 0.0001)