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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: Melatonin synergizes BRAF-targeting agent vemurafenib in melanoma treatment by inhibiting iNOS/hTERT signaling and cancer-stem cell traits

Fig. 4

Melatonin enhanced the vemurafenib-induced inhibition of INOS expression by inhibiting NF-κB signaling pathway. (a). The expression level of iNOS and p-P65 protein were analyzed by Western blot in human melanoma cells treated with the indicated doses of vemurafenib (VE) (2.5 μM) and melatonin (MT) (1.0 mM) for 48 h. (b). Cell viability was analyzed by MTT assay in melanoma cells treated with vemurafenib (2.5 μM) combined with or without MT (1.0 mM) for 24 h after pretreatment with the iNOS targeting siRNA for 48 h. (c). The expression of p50/p65 was determined from nucleus extracts prepared from melanoma cells by Western blotting. (d). The subcellular localization of p50 and p65 and their co-localization in human melanoma cells treated with 2.5 μM vemurafenib (VE) and 1.0 mM melatonin (MT) for 48 h were examined by confocal microscopy. Cells with typical morphology were presented from more than 100 cells at each experiment. (e). The streptavidin-biotin pulldown assay was performed to analyze the binding of P65 protein to iNOS promoter in melanoma cells with the indicated treatment. (f). Binding of p65 to the iNOS promoter in chromatin structure by ChIP assay. IgG, a negative control for ChIP in melanoma cells with the indicated treatment. (g). Human melanoma cells were treated with 2.5 μM vemurafenib (VE) and 1.0 mM melatonin (MT). At 48 h after treatment, the IKKβ, p-IKKβ, IκBα and p-IκBα proteins were analyzed by Western blotting. (h). Vemurafenib (VE) combined with or without 1.0 mM melatonin (MT) followed by NF-κB inhibitor treatment and then iNOS expression and cell viability was respectively analyzed Western blot. (i). MTT assay in melanoma cells treated with NF-κB Activation Inhibitor followed by the treatment of vemurafenib (VE) combined with or without 1.0 mM melatonin (MT). Each data point was calculated from three triplicate groups and the data is presented as the mean ± SD. *P < 0.05, significant difference between treatment group and control group

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