Figure 5.

Dynamic change of AHR expression and allele-preferential AHR binding to rs117132860-G in melanocytes upon dioxin and UVB exposure

(A) AHR expression normalized to GADPH was measured by Taqman assay in C87 human melanocytes before and after TCDD treatment (individual datapoints, mean, and SEM are plotted).

(B) AHR immunoblotting indicates a dynamic change of AHR protein nuclear localization after TCDD treatment.

(C) GAPDH-normalized AHR transcription is increased after UVB exposure in C87 melanocytes (individual datapoints, mean, and SEM are plotted).

(D) Nuclear translocation of AHR is observed after UVB exposure via immunoblotting in C87 melanocytes.

(E—H) AHR binding to rs117132860 measured by CHIP assay increased after (E) TCDD treatment in C197 melanocytes and (G) UVB exposure in C87 melanocytes. ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001 (individual datapoints, mean, and SEM are plotted). A genotyping assay with AHR CHIP DNA shows the enhanced binding of AHR to the rs117132860-G allele after (F) TCDD treatment and (H) UVB exposure in C87 melanocytes (individual datapoints, mean, and SEM are plotted).

(I) A genotyping assay of rs17779352, a proxy SNP for rs117132860 located in in the AHR coding sequence, indicates that the ratio of AHR expression from the melanoma-protective rs117132860-G/rs17779352-C allele significantly increased after UVB exposure relative to the rs117132860-A/rs17779352-T allele at 24 h.

All experiments except for those shown in (I) were done in both C87 and C197 human melanocytes with a total of four biological replicates each. The experiment set shown in the figure is a representative one with p value calculated from a two-way paired Student’s t test (A, C, E, and F) or two-way ANOVA (G and H), and the mean with SEM is plotted. The proxy SNP genotyping experiment (I) was only performed in C87 cells, which are heterozygous for both rs117132860 and rs17779352; the violin plot shows the combination of four experiments of three replicates each, and all points are shown.