Fig 2: Gcm1 competes with ß-catenin for binding to TCF4 and is associated with Wnt target genes expression.a ChIP-qPCR analysis of Gcm1, ß-catenin and TCF4 enrichment in Wnt-responsive element (WREs) of Axin2 in control and folate-deficient mESCs. NEG negative control, TSS transcription start site. Right panel: the locations of the WRE and NEG in the promoter. b ChIP-qPCR analysis of Gcm1, ß-catenin, and TCF4 enrichment in the WREs of Bcl9l in control and folate-deficient mESCs. Right panel: the locations of the WREs and NEG in the promoter. c ChIP-qPCR analysis of Gcm1, ß-catenin, and TCF4 enrichment in the WREs of Isl1 in control and folate-deficient mESCs. Right panel: the locations of the WREs and NEG in the promoter. d Functional clustering of genes associated with Gcm1 ChIP-peaks in mESCs with folate deficiency (the top 10 categories are shown). The X-axis represents the log10 value of the binomial raw P value. e Pathway analysis of Gcm1 ChIP-peaks in mESCs with folate deficiency (the top 10 categories are shown). Gcm1 binding pathway enrichment was determined using PANTHER analysis. The X-axis represents the enrichment factor, the Y-axis represents the binomial raw P value, and the size of each circle represents the number of genes. f Representative examples of Ctla4 and Edn1 chromatin binding. A segment of chromosome 1 demonstrates the decreased binding of Clta4 to a TBE in folate-deficient compared to normal mESCs. A segment of chromosome 13 demonstrates the decreased binding of Edn1 to a TBE in folate-deficient group compared to normal mESCs. g ChIP-qPCR analysis of Clta4 and Edn1 in mESCs with folate deficiency. *P = 0.015 (Clta4) and *P = 0.022 (Edn1). h The mRNA expression of Clta4 (J) and Edn1 (K) in mESCs with folate deficiency. *P = 0.015 (Clta4) and *P = 0.04 (Edn1). i Correlation analysis between ChIP density and the mRNA expression of Clta4 and Edn1. The correlation coefficients for Clta4 is 0.739 (P = 0.003), and for Edn1 is 0.45 (P = 0.002). j Correlation of the tag densities (Y-axis) with the transcription levels of genes from -3 kb to +3 kb (X-axis). All the genes were divided into five groups based on their transcription levels from high (dark blue) to low (orange). The mean-normalized ChIP-seq densities with 1-bp resolution are plotted within a 6-kb region flanking the TSS or the TTS (transcription termination site). Data a–c, g–i represent the mean ± SEM (n = 3). The p value was calculated by Student’s t-test, ns was for no significance, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: CYP2E1-inhibited Wnt/ß-catenin signaling by ROS. A ROS level in control and CYP2E1-overexpressing HCC cells detected by fluorescence probe and the change in ROS level after treatment with the CYP2E1 inhibitor CMZ (40 µM) or the antioxidant NAC (10 mM) for 6 h in the indicated cells. B The OD450 value of MHCC-97H-CYP2E1, SMMC-7721-CYP2E1 and the respective control cells after treatment with NAC (10 mM) at the indicated time points. C Representative images and statistical analysis (right panel) of scratch wound assays of MHCC-97H-CYP2E1, SMMC-7721-CYP2E1 and the respective control cells after treatment with NAC (10 mM) (×100). D Representative images and statistical analysis (down panel) of transwell assays of MHCC-97H-CYP2E1, SMMC-7721-CYP2E1, and the respective control cells after treatment with NAC (10 mM) (×100). E After treatment with the CYP2E1 inhibitor CMZ (40 µM) or the antioxidant NAC (10 mM) for 6 h, the protein levels of Dv1-2, ß-catenin, TCF4 and c-Myc in MHCC-97H-CYP2E1, SMMC-7721-CYP2E1 and the respective control cells were evaluated by western blot. Data in 5B, 5C and 5D are shown as mean ± sd (n = 3–6 in each assay). *P = 0.05, **P = 0.01, ***P = 0.001 vs. CYP2E1

Fig 4: CYP2E1 inhibits Wnt/ß-catenin signaling via reducing the Dvl2 protein level. A Luciferase activity of TOPFlash in MHCC-97H-CYP2E1, SMMC-7721-CYP2E1 and their respective control cells. Data are shown as mean ± sd (n = 3 in each assay). *P = 0.05, **P = 0.01 vs. control cells. B Protein level of Dvl2, ß-catenin, TCF4, LEF1 and c-Myc (Wnt/ß-catenin signaling-related molecules) in control and CYP2E1-overexpressing HCC cells measured by western blot. C The protein expression of CYP2E1, Dvl2, ß-catenin, and c-Myc in control and CYP2E1-overexpressing HCC cells at 6 h after treatment with the CYP2E1 inhibitor CMZ (40 µM). D The OD450 value of MHCC-97H-CYP2E1, SMMC-7721-CYP2E1 and the respective control cells after treatment with the CYP2E1 inhibitor CMZ (40 µM). E Representative images and statistical graphs (right panel) of transwell assays for the indicated cells after treatment with the CYP2E1 inhibitor CMZ (40 µM) (×100). F Representative images and statistical graphs (right panel) of scratch wound assays of MHCC-97H-CYP2E1, SMMC-7721-CYP2E1 and the respective control cells after treatment with the CYP2E1 inhibitor CMZ (40 µM) (×100). Data in 3D, 3E and 3F are shown as mean ± sd (n = 3–6 in each assay). *P = 0.05, **P = 0.01, ***P = 0.001 vs. CYP2E1

Fig 5: Effect of FoxM1 silencing and forced expression in EOC cell linesSilencing of FoxM1 down regulates the expression of ß-catenin and their downstream targets as well as inhibits tumorigenesis in nude mice. (A) Silencing of FoxM1 inhibits ß-catenin and associated downstream targets. EOC cells were transfected with scrambled siRNA and FoxM1 siRNA (50 and 100 nM). After 48 hours, cells were lysed and proteins were immunoblotted with antibodies against FoxM1, active-ß-catenin, ß-catenin, TCF4, Cyclin D1, cMYC, uPAR, VEGF, MMP-9, MMP-2 and ß-actin. (B) Representative images of fluorescence immunostaining for FoxM1 and ß-catenin in EOC cells after post-transfection with FoxM1 siRNA. (C–E) Silencing of FoxM1 inhibits tumorigenesis in nude mice. OVCAR3 cells were transfected with scrambled siRNA and FoxM1 siRNA (100 nM). Cells (2 × 106 cells) were injected into the flanks of 6-week old nude mice (n = 5). Solid tumors in the FoxM1 silenced OVCAR3 cells injected mice were significantly smaller than those mice injected with OVCAR3 alone or OVCAR3 transfected with scramble siRNA. Silencing of FoxM1 significantly reduced (C) tumor volume, and (D) tumor weight in the mice injected with FoxM1 silenced SKOV3 cells. (E) Representative tumor images of xenografts. (F) Tissue lysates were immuno-blotted with FoxM1, active-ß-catenin, ß-catenin, TCF4, and ß-actin antibodies. (G–H) Forced expression of FoxM1 induces ß-catenin and associated downstream targets. (G) Representative images of fluorescence immunostaining for FoxM1 and ß-catenin in OVTOKO cells after post-transfection with FoxM1 plasmid. (H) OVTOKO cells were transfected with either empty vector or FoxM1 plasmid for 48 hours. Proteins were isolated and were immuno-blotted with antibodies against FoxM1, active-ß-catenin, ß-catenin, TCF4, Cyclin D1, cMYC, uPAR, VEGF, MMP-9, MMP-2 and ß-actin.