Prevention of estrogen-mediated suppression of OGG1 by antioxidants is NRF2-dependent. (A) Real-time PCR data (normalized to internal control cyclophilin) are presented as a bar graph to show expression levels of NRF2 mRNA in mammary tumors and mammary tissues of rats treated with E2, Vit C or BHA in presence or absence of E2 for 240 days. These data are reported as an average of fold change values obtained from at least 5 different animals ± SEM, compared to age-matched control mammary tissues. ‘*’ indicates p value <0.05 compared to respective control mammary tissues. (B) Western blot analyses were carried out to determine protein expression levels of NRF2 in mammary tumors and mammary tissues of rats treated with E2, Vit C, Vit C + E2, BHA and BHA + E2 for 240 days. The bar graph represents NRF2 protein fold change (mean ± SEM) in mammary tumors and mammary tissues from at least 5 different animals compared to age-matched control mammary tissues. ‘*’ indicates p value <0.05 compared to respective, age-matched control mammary tissues. (C) MCF-10A cells were transfected with 20 nmol/L of scrambled siRNA or siNRF2 for 48 h and western blot analysis was carried out using NRF2 antibody. Same membrane was reprobed with OGG1 and α-Tubulin antibodies. Concomitant with a decrease in NRF2 protein expression, there is a corresponding decrease in OGG1 protein expression. (D) MCF-10A cells were treated with E2 (10 nM), Vit C (1 mM), BHA (250 μM) or vehicle for 45 min, fixed with formaldehyde, cross-linked, and the chromatin sheared. The chromatin was immunoprecipitated with NRF2 antibody. NRF2 binding to OGG1 promoter was analyzed by real-time PCR with specific primers for the human OGG1 ARE region. The ARE region of the OGG1 promoter was also amplified from purified soluble chromatin before immunoprecipitation to show input DNA. Representative ChIP agarose gels and real-time PCR Ct values from three independent experiments are shown. (ND = not detected).