Fig. 5

Combination of vemurafenib and melatonin inhibited cancer stem cell traits by down-regulating hTERT in melanoma cells. Human melanoma cells were exposed to vemurafenib (VE) (2.5 μM) with or without melatonin (MT) (1.0 mM) for 48 h. (a). The representative images of tumor sphere formation of melanoma cells with indicated treatment. (b). CD44 expression on the surface of melanoma cells was analyzed by FACS. (c). The expression of CSC-related markers Epcam, CD44, c-kit and Oct4 were determined by western blot in A375 and SK-mel-28 cells with the indicated treatment. (d). The expression of hTERT-p-MSK1-p65 pathway were determined by western blot in A375 and SK-mel-28 cells with the indicated treatment. (e). The representative images of tumor sphere formation of melanoma cells treated with DMSO or vemurafenib (2.5 μM) combined with MT (1.0 mM) for 24 h after pretreatment with the hTERT targeting shRNA. (f). Melanoma cells were co-treated with the plasmids of hTERT promoter driven-luciferase and vemurafenib (VE) (2.5 μM) with or without melatonin (MT) for 48 h followed by a dual-luciferase assay. The relative luciferase intensity per mg protein was calculated in the treated cells. (g). The streptavidin-biotin pulldown assay was performed to analyze the binding of P65 protein to hTERT promoter in melanoma cells with the indicated treatment. (h). Binding of p65 to the hTERT promoter in chromatin structure by ChIP assay. IgG, a negative control for ChIP in melanoma cells with the indicated treatment. The data are presented as the mean ± SD of three separate experiments. ^*P < 0.05, significant differences between treatment groups and DMSO control groups