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MiTF-S73A is less potent in activating p21WAF1/CIP1 transcription. A, p21WAF1/CIP1 and p27KIP1 protein accumulation in A375 cells expressing MiTF-WT, MiTF-S73A and GFP were analyzed by western blot. B, transcripts of p21WAF1/CIP1 and MiTF were analyzed by qRT-PCR in the above cells, α-tubulin was the reference for both genes. C, p21WAF1/CIP1 promoter reporter analysis in cells co-transcfected with MiTF-WT or MiTF-S73A mutant constructs. D, p21WAF1/CIP1 protein accumulation decreased when MiTF phosphorylation was inhibited. NHMs were treated with 20 μM of U0126 for 24 hours and collected for western blot analysis. E, knockdown MiTF led to decreased p21WAF1/CIP1 expression. Quantitative RT-PCR (left) and western blot (right) analysis of p21WAF1/CIP1 expression in control SK-Mel-28 cells (Mel-28), cells transduced with empty lentivirus vector pGIPZ (GIPZ), and cells transduced with lentivirus carrying MiTF shRNA constructs (Mish1 and Mish2). Again α-tubulin was used as a loading control. F, top: p27KIP1 protein accumulation after UVC in A375 cells expressing MiTF-WT, MiTF-S73A and GFP; bottom: p21WAF1/CIP1 protein accumulation after UVC in A375 cells expressing MiTF-WT, MiTF-S73A and GFP. The p53 served as a positive control for UVC radiation, α-tubulin served as a loading control. The western was repeated three times and a representative blot is shown; G, the p21 protein levels in the western blot were quantified by a densitometry, normalized to α-tubulin levels and then normalized to that in cells without irradiation and graphed. H, qRT-PCR analysis of p21WAF1/CIP1 mRNA accumulation after UVC in A375 cells expressing MiTF-WT or MiTF-S73A.