Fig. 6

Positive correlation between RHBDD1 and ZEB1 from CRC cells to tumor tissues from patients. a. Q-PCR analysis of ZEB1 and RHBDD1 mRNA levels in HCT-116 cells with knockdown of RHBDD1. Ctrl, control; Si-1#, Si-RHBDD1–1#; Si-2#, Si-RHBDD1–2#; **, P < 0.01; ***, P < 0.001. b. Immunoblot analysis of ZEB1 and RHBDD1 protein levels when in HCT-116 cells with knocked down, over-expressed, knocked out or rescued RHBDD1 expression. Si-pool, mixture of Si-RHBDD1–1# and Si-RHBDD1–2# in a ratio of 1:1; OE, over-expression. WT, wild-type; KO, cas9 knocked out; MT + Ctrl, mutant cell line infected with lentivirus-control; MT + OE, mutant cell line infected with lentivirus-RHBDD1. c. HCT-116 cells were co-transfected with si-RHBDD1 miRNA or control miRNA and empty vector, S552D-β-catenin or S675D-β-catenin plasmids simultaneously, 48 h after which immunoblot analysis of ZEB1, RHBDD1 and β-catenin was performed. d. Wild-type HCT-116 cells and RHBDD1 mutant HCT-116 cells were transfected with empty vector, S552D-β-catenin or S675D-β-catenin constructs and then subjected to immunoblot analysis of ZEB1, RHBDD1 and β-catenin. b, c, d. Bands were quantified with ImageJ software. e. Immunoblot analysis of ZEB1 and RHBDD1 expression levels in 8 CRC tumor tissues, Tubulin was used as a loading control. Bands were quantified using ImageJ software. The correlation between ZEB1 and RHBDD1 protein levels were calculated using GraphPad Prism 5 software, the dots in the data represent the expression levels of RHBDD1 and ZEB1 relative to tubulin in the indicated tissues. f. Images of tissues showing the levels of ZEB1 and RHBDD1 protein expression in the tissue microarray analysis. g. Correlation between the levels of ZEB1 and RHBDD1 protein expression in 71 tissues of the tissue microarray analysis. The protein expression levels were evaluated using an immunostaining score as indicated in the Methods section. Numbers beside the dots represent the sample sizes with indicated immunostaining score of ZEB1 and RHBDD1. The dots’ areas are coincident with the sample sizes