Figure 2: MC1R expression regulates the formation of mutagenic photoproducts.
From: Germline MC1R status influences somatic mutation burden in melanoma
(a) Shown are HPMs stably expressing control shRNA (shScr) or multiple independent shMC1Rs targeting MC1R (#1, #2, #3). Cells were pre-incubated with 1 μM α-MSH for 30 min, then exposed to 100 J m−2 UVB light, and harvested 3 h later for Western blot analysis. (b) HPM cells stably expressing shMC1R#1 were irradiated with different doses of UVB light as indicated. Cells were collected at 24 h after UVB light irradiation and viability assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (c) HPMs stably expressing an shRNA against MC1R (shMC1R#1) or a scrambled control (ShScr) were transfected with 2 μg of UV damaged pGL3 luciferase expression vector and 0.5 μg pRL Renilla Luciferase control reporter vector before the host cell reactivation assay28,32 (Methods section; d,e). HPMs stably expressing an shRNA against MC1R (shMC1R#1) or a scrambled control (ShScr) were irradiated with 100 J m−2 UVB light and then collected at the different time points indicated. Genomic DNA was extracted and photoproducts were detected by ELISA. Cyclobutane pyrimidine dimer (CPD) (d) or 6–4 pyrimidine photoproduct (6–4PP) (e) antibodies were used (Methods section). These data were compiled from three separate experiments performed in triplicate. Significance (*P<0.05) was calculated by using the unpaired two-tailed Student’s t-test comparing the means (shScr vs shMC1R#1) of the three experiments. Error bars represent s.d.
