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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: RHBDD1 promotes colorectal cancer metastasis through the Wnt signaling pathway and its downstream target ZEB1

Fig. 4

RHBDD1 influences Wnt signaling activity by regulating the phosphorylation of ser552 and ser675 of β-catenin. a. Immunoblot analysis of RHBDD1, total β-catenin and indicated phosphorylated β-catenin in the isolated cytosolic and nuclear lysates from HCT-116 cells transfected with RHBDD1 Si-RNA and control Si-RNA. GAPDH was used as the cytosol marker, and Lamin A/C was used as the nuclear marker. Ctrl, control; Si-pool, mixture of Si-RHBDD1–1# and Si-RHBDD1–2# in a ratio of 1:1; C, cytosol; N, nucleus. Bands were quantified with ImageJ software. b. TOP/FOP flash reporter assay of HCT-116 cells stimulated with CHIR99021. DMSO was used as the solvent control. HCT-116 cells were transfected with Control and RHBDD1 Si-RNA, and 48 h later, cells were stimulated with 100 ng/ml CHIR99021 or solvent control DMSO dissolved in serum free culture medium for 24 h, then cells were harvested for luciferase analysis. c. Immunoblot analysis of isolated cytosolic and nuclear protein fractions from HCT-116 cells stimulated with CHIR99021. d. Wild-type HCT-116 cells and HCT-116 cells stably expressing mutant RHBDD1 were transfected with empty vector, S552D-β-catenin or S675D-β-catenin, and the TOP/FOP flash reporter assay was performed. Immunoblot analysis of RHBDD1 and β-catenin in transfected HCT-116 cells. Tubulin was used as a loading control. WT, wild-type; MT, mutant; EV, empty vector; 552D, S552D mutant construct; 675D, S675D mutant construct. e. HCT-116 cells stably expressing mutant RHBDD1 were transfected with empty vector, S552D-β-catenin or S675D-β-catenin and subjected to cell migration and invasion assays. Images of representative wells of cell migration and invasion assay were shown, cells were counted and calculated. Immunoblot analysis of RHBDD1 and β-catenin in transfected HCT-116 cells was performed. a, c, d, e. Bands were quantified with ImageJ software. The experiments in b, d, and e were performed in triplicate. All data are mean ± SEM.*, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant

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