Fig 2: Methylation level of WIF1 in CS patient tissues. (A) Methylation levels of WIF1 in each specimen are presented. (B) Distributions are shown for 43 CS tumor samples.

Fig 3: 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)

Fig 4: NRP2 expression is regulated by Wnt signaling pathway. A, Genechip® microarray showed the down-regulated expression of NRP2 in Wnt antagonist sLRP5 transfected osteosarcoma Saos-2 cells. VEGF expression is intact. Real time PCR (B) and western blot (C) with accompanying densitometric assay (D) confirmed the down-regulated mRNA and protein level of NRP2 in Wnt antagonist sLRP5 transfected Saos-2 cells, while VEGF level remains intact. E, western blot and accompanying densitometry showed the down-regulated NRP2 expression in additional Wnt antagonist DKK3 transfected Saos-2 cells, 143B cells and U2-OS cells, and Wif-1 transfected 143B cells. F, Chromatin immunoprecipitation (ChIP) assay verified the binding of TCF4 on the five binding sites in NRP2 promoter region. G, schematic representation of the binding sites of TCF/LEF on the 3-kb promoter region of NRP2 genes.

Fig 5: Kaplan-Meier curves of overall survival and progression-free survival of different quartiles of WIF1 methylation level in CS. (A) Kaplan-Meier curves of OS showed a significant difference across the Q1, Q2, Q3, and Q4 groups (p = 0.016). (B) Kaplan-Meier curves of PFS showed a weak difference among these four groups, but was not significant (p = 0.094). (C) OS of Q1 group was significantly shorter than the Q2–4 group (p = 0.008). (D) PFS of Q1 group was significantly shorter than the Q2–4 group (p = 0.023). Q1 represents the “high-methylation-level group” and Q2, Q3, and Q4 collectively represent the “low-methylation-level group”.