Fig. 1

Indirubin inhibits the expression of DNMT1, restores wif-1 expression, and inhibits wnt/β-catenin signal pathway. (a): The expression of wif-1 was promoted and the expression of DNMT1, Frizzled2, Frizzled5, and phosphorylation β-catenin was suppressed after treatment with different concentrations (0.04 μM, 0.2 μM, and 1 μM) of indirubin for 48 h compared with the negative control group in HaCaT cells by Western blotting using GAPDH as an internal control. (b): The mRNA expression levels of related proteins after treated with different concentrations (0.04 μM, 0.2 μM, and 1 μM) of indirubin. The mRNA expression level of wif-1 was promoted, and the mRNA expression levels of DNMT1, Frizzled2, Frizzled5, and β-catenin were suppressed compared with the negative control group in HaCaT cells by qRT-PCR. (c): WIF-1 promoter methylation level decreased after treated with low (0.04 μM), medial (0.2 μM), and high(1 μM) concentrations of indirubin while abnormal methylation was observed in the negative control group. (d): The silencing of DNMT1 suppresses wif-1 promoter hypermethylation in HaCaT cells, likewise, wif-1 promoter hypermethylation was suppressed after treated with indirubin(1 μM) together with si-NC, further, wif-1 promoter hypermethylation was significantly suppressed after treated with indirubin(1 μM) together with si-DNMT1, relative to the si-NC group (Fig.1e). (e): The overexpression of DNMT1 significantly promoted wif-1 promoter methylation level in HaCaT cells, nevertheless,wif-1 promoter methylation level was suppressed after treated with indirubin(1 μM) together with control,wif-1 promoter methylation level was promoted after treated with indirubin(1 μM) together with DNMT1, relative to the control group. (f): The protein expression of wif-1 was encouraged after treated with different concentrations (0.04 μM, 0.2 μM, and 1 μM) indirubin compared with the negative control group in HaCaT cells by ELISA. (g): The mRNA expression of DNMT1 was suppressed after treated with different concentrations (0.04 μM, 0.2 μM, and 1 μM) of indirubin compared with the negative control group in HaCaT cells by ELISA. (d): A: si-NC, B: si-DNMT1, C:si-NC + indirubin, D:si-DNMT1 + indirubin. (e): A: Ctrl, B: DNMT1, C: Ctrl+indirubin, D: DNMT1+ indirubin. *P < 0.05, **P < 0.01, ***P < 0.001. U: unmethylated; M: methylated (N = 3)