Fig. 4

Inhibition of AP-2α expression will inhibit the transformation of CD133-positive HCC cells induced by POSTN; a Western Blot was used to test CD133, AP-2α, and POSTN expression in Hep3B cell after transfected with control or p-POSTN plasmid, followed by AP-2α shRNA to target AP-2α. b CD133, AP-2α, and POSTN protein expression was carried out by rescue experiment targeting POSTN and then recovered AP-2α expression in POSTN-expressing LM3 cell. c Flow Cytometry Analysis was used to quantify CD133-positive hepatoma cells number after transfected with control or p-POSTN plasmid, followed by AP-2α shRNA to target AP-2α as well as targeting POSTN and then recovered AP-2α expression in POSTN-expressing HCCLM3 cell. d Immunofluorescence dual staining was used to verifiy that after down-regulation of POSTN expression in hepatoma cells, restoring AP-2α (Green) expression was still effective in up-regulating CD133 (Red) expression induced by POSTN. Scale bar, 10 μm. e The reporter plasmid map of double luciferase contains the promoter region sequence of CD133 gene, which can be matched with AP-2α partial base complementary. f The Luciferase Report experiment confirmed that inhibition of AP-2α expression would effectively inhibit the luciferase activity of CD133 promoter caused by POSTN, and recovery of AP-2α expression in the POSTN down-regulated HCC cells would promote the luciferase activity of CD133 promoter. g ChIP experiment further confirmed the direct binding of AP-2α to CD133 promoter region in LM3 cells, IgG and AP-2α in parenthesis stated the targeting of antibody used in chromatin immunoprecipitation assay; h-k Clone formation (h), Invasion (i), Sphere formation ability (j) and viability (k) of hepatoma cells were measured after transfected with control or p-POSTN plasmid, followed by AP-2α shRNA to target AP-2α as well as targeting POSTN and then recovered AP-2α expression in POSTN-expressing LM3 cell. l Inhibition of AP-2α expression will effectively inhibit the high tumorigenicity of POSTN in HCC cells。Data were represented as mean + SEM from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001