Fig. 3

Depression of RHBDD1 attenuates Wnt signaling pathway activity and influences β-catenin protein expression. a. Top 10 signaling pathway based on KEGG enrichment analysis of signal transduction pathway sub-categories. Count, differential gene count in indicated pathway; padj, adjusted P value of indicated pathway; GeneRatio, differential gene count in indicated pathway versus total differential gene count. RHBDD1 was knocked down with Si-RHBDD1-pool, mixture of Si-RHBDD1–1# and Si-RHBDD1–2# in a ratio of 1:1. b. Dual-luciferase reporter assay of Wnt signaling pathway. The TCF/LEF binding regions were used as indicated in the Methods section. Ctrl, control; Si-1#, Si-RHBDD1–1#; Si-2#, Si-RHBDD1–2#; ***, P < 0.001. c, d. TOP/FOP flash reporter assay in cells with RHBDD1 knockdown (c), mutant RHBDD1 (c) and rescued wild-type RHBDD1 (d), respectively. The TOP flash plasmid or FOP flash plasmid and pRL-TK plasmid were used as indicated in the Methods section. Ctrl, control; Si-pool, mixture of Si-RHBDD1–1# and Si-RHBDD1–2# in a ratio of 1:1; WT, wild-type; MT, mutant; MT + Ctrl, mutant cell line infected with lentivirus-control; MT + OE, mutant cell line infected with lentivirus-RHBDD1. The experiments in b, c, and d were performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001. e. Immunoblot analysis of RHBDD1, total β-catenin and phosphorylated β-catenin in cells with knocked down and over-expressed RHBDD1. Tubulin was used as a loading control. Bands were quantified with ImageJ software