Fig 2: Antagonism toward AHR binding at the BRCA1 promoter by GEN. Results show the relative binding of AHR at the BRCA1 promoter from chromatin immunoprecipitation (ChIP) assays in (A) HCC38 cells treated with GEN and various AHR antagonists; and (B) MCF7 cells treated with estradiol (E2) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of GEN and GAL. Relative binding was determined as the percent input (ChIP/Input*100) from treated samples normalized to VEH (A) or E2 (B) controls. Bars represent sample means ± SEM from =3 biological replicates from individual experiments. Means with different letters (a > b) or asterisks (*) indicate significance (*, p < 0.05).

Fig 3: Rescue of BRCA1 expression is linked to CpG demethylation in TNBC cells. The average ratio of mBRCA1/umBRCA1 in HCC38 cells treated for 96 h with (A) GEN or (B) GAL. Bars represent sample means ± SEM from =3 biological replicates from individual experiments. Asterisks denote significance (*, p < 0.05).

Fig 4: Lifetime exposure to genistein (GEN) decreases basal Brca1 cytosine-guanine dinucleotide (CpG) methylation and aryl hydrocarbon receptor (AHR) activity in mouse offspring mammary tissue. Mice were exposed to diets containing 0, 4, or 10 ppm GEN starting at conception and through nursing, weaning, and adulthood. (A) Brca1 CpG methylation in a mammary gland of post-natal day 50 (PND50) female offspring. Bars represent the mean ratios of methylated Brca1 to unmethylated Brca1 amplicons ± SEM from =7 individual animals. (B) Cyp1b1 mRNA expression in PND50 mammary glands. Bars represent mean RQ ± SEM from =7 individual animals. Means with different letters (a > b > c) or asterisks (**, p < 0.01) indicate statistical significance (p < 0.05).

Fig 5: Genistein rescues BRCA1 protein levels in TNBC cells with CpG hypermethylated BRCA1. Bands are immunocomplexes of BRCA1 and internal standard GAPDH in HCC38 cells treated with (A) GEN, (B) a-naphthoflavone (NF), (C) CH-223191, and (D) galangin (GAL). Images are representative blots from =3 individual experiments. For densitometry histograms, bars represent mean RQ ± SEM from =3 biological replicates from individual experiments. Asterisks indicate significant differences (*, p < 0.05; ***, p < 0.001).