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?1d; P?this website mechanism in the action of SIRT2, we measured the role of SIRT2 in critical processes involved in skin tumorigenesis, including DNA damage repair, apoptosis and proliferation in normal human epidermal keratinocytes (NHEK), transfected with negative control (siNC) or siRNA targeting SIRT2 (siSIRT2) (Fig. S1a�Cd). We found that SIRT2 had no effect on the repair of UVB-induced cyclobutane pyrimidine dimers (CPD), the major DNA lesions (Fig. S1a) or the levels of proteins involved in UVB-induced DNA damage repair, including XPC, DDB1 or DDB2 (Fig. S1b). SIRT2 did not significantly affect p53 acetylation or p53 levels (Fig. S1b), UVB-induced apoptosis or cell proliferation (Fig. S1c�Cd). To determine the molecular function of SIRT2, we carried out a microarray analysis of gene expression in siNC- and siSIRT2-transfected NHEK cells. Among the genes that were up- or downregulated by SIRT2 knockdown (Table S1), several were genes involved in keratinocyte differentiation. SIRT2 inhibition increased the expression of keratin 19 (K19) and keratin 15 (K15), while it decreased the expression of transglutaminase 1 (TGM1) (Fig.?2a). Immunoblot analysis indicated that SIRT2 knockdown increases K19 protein levels (Fig.?2b), suggesting that SIRT2 plays a role in keratinocyte differentiation. Galunisertib To determine the role of SIRT2 in keratinocyte differentiation induced by confluence, we measured the levels of SIRT2, K19 and the differentiation markers involucrin and p27 in NHEK cells at subconfluence, confluence and postconfluence. As compared with their subconfluent cells, while SIRT2 was upregulated, K19 was downregulated in NHEK cells transfected with siNC in confluence for 1 and 3?days, in parallel with RhoC the upregulation of p27 and involucrin (Fig.?2c). Knockdown of SIRT2 delayed upregulation of p27 and involucrin after confluence, and increased K19 levels before and after confluence (Fig.?2c). To determine the role of SIRT2 in keratinocyte differentiation and stemness, we assessed the difference in the levels of K19, the differentiation marker Loricrin, the stem cell marker CD34 and K15, and the number of Ki67-positive cells in normal skin and tumors between SIRT2 WT and KO mice. As compared with SIRT2 WT mice, in SIRT2 KO mice, K19 and K15 were increased in both normal skin and tumors (Fig.?2d), while Loricrin was decreased. In tumors but not in normal skin, SIRT2 deletion increased CD34 expression and number of K67-positive cells (Fig.?2d). These results indicate that SIRT2 promotes keratinocyte differentiation and that SIRT2 loss promotes tumor cell stemness and proliferation.
