Fig. 4
Modulation of AHR activity affects AHR binding to Cyp1b1 and Sox2 promoters and SOX2 protein production. (a) Hs578T cells were treated with vehicle, 10 μM AHR inhibitor CH223191, 0.5 μM FICZ, or 0.5 μM FICZ + 10 μM AHR inhibitor CH223191 for 48 hours and ChIP assays were performed with human AHR-specific antibody and Cyp1b1- or Sox2-specific promoters as described in the Materials and Methods. Data are presented as mean fold-change ± standard error, IgG control n = 4, vehicle n = 5, CH223191 n = 4, FICZ n = 5, FICZ + CH223191 = 4. An asterisk indicates a significant increase relative to vehicle controls, *P <0.05. A pound sign indicates a significant decrease relative to vehicle controls, # P <0.05. A cross indicates a significant increase relative to IgG controls, + P <0.005. A caret sign indicates a significant decrease relative to FICZ treatment, ^P <0.05. Relative positions of putative AHR response elements and amplified fragments are represented in the embedded map. (b) SUM149 or (c) MCF-7 cell cytoplasmic and nuclear protein extracts were probed for SOX2 protein expression following treatment with vehicle or AHR agonists: 0.5 μM FICZ, 10 μM B(a)P or 1 nM TCDD. C = cytoplasmic extract, N = nuclear extract. The number above each band indicates fold-change from naïve after normalization to loading control, based on ImageJ densitometry analysis. (d) Hs578T cells were transfected with a CMV promoter-driven Sox2 plasmid and ALDH activity assayed 48 hours later. Data from four independent experiments are presented as percent ALDHhigh ± standard error. Asterisk indicates a significant increase in the %ALDHhigh cells, *P <0.005