Fig. 7
Downregulation of overexpressed genes with hypermethylated gene body CGI in Alb-R26Met HCC cells interferes with their tumorigenic properties both in vitro and in vivo. a Western blots showing SCN8A, ACTN1, SRD5A2, NFkB2 and NEURL1B protein levels in stable clones established after transfection of Alb-R26Met HCC14 cells with plasmids carrying a shRNA sequence targeting the corresponding gene. Protein levels were compared to control cells (ctr). ACTIN was used as a loading control in all western blots. The asterisk indicates nonspecific bands detected using anti-SRD5A2 antibodies. b–e Biological assays to assess functional properties of Alb-R26Met HCC14 cells carrying a shRNA sequence targeting candidate genes. Effects were compared to HCC14 cells either untransfected or transfected with a control shRNA (shCtrl). b Graph reporting the number of colonies formed in anchorage-independent growth assays using 2 different shRNA targeting sequences for each candidate gene. Note a decrease in colony number formation of cells with downregulated candidate genes compared with control cells. c Graphs reporting the number (left) and the size (right) of colonies formed in anchorage-dependent growth assays. Whereas no significant changes in colony numbers were detected, note a significant decrease in colony size when the candidate gene is downregulated. d Graph reporting number of spheres formed in tumour sphere assays. Note that downregulation of candidate genes significantly reduces sphere number formation. e Graph reporting the tumour volume of mice injected either with Alb-R26Met HCC14 control cells or with Alb-R26Met HCC14 cells carrying a shRNA sequence targeting candidate genes. Note that downregulation of candidate genes significantly interferes with the in vivo tumorigenic properties of Alb-R26Met HCC14 cells. Significant differences between groups are indicated on the top. *P < 0.05, **P < 0.01, ***P < 0.001 (nd: no determined)
