Fig. 2

RNF152 is a transcriptional target of FoxO1 in HCC. a The mRNA levels of GSTP1 and RNF152 in HepG2 cells with or without 10 μM 5-Aza-CdR treatment were determined by real-time PCR. ***P < 0.001. b The correlation between RNF152 and FoxO1 mRNA in the TCGA HCC dataset through the analysis of the GEPIA website. c Schematic diagram shows human RNF152 gene promoter and putative FoxO1 binding sites. TSS: transcription start site. IRE: insulin response element. The primer pairs for Chip assay were also listed. d pGL4.15-Con or pGL4.15-RNF152 plasmids were co-transfected with either empty vector (EV) or FoxO1 into 293 T cells for 36 h. The luciferase activity was then measured. Overexpression of FoxO1 activated RNF152 promoter-driven luciferase activity. **P < 0.01. e HepG2 cells were transfected with EV or FoxO1 for 36 h. The mRNA levels of FoxO1 and RNF152 were determined by real-time PCR. ***P < 0.001. f HepG2 cells were transfected with con-shRNA or FoxO1-shRNA for 36 h. The mRNA and protein levels of RNF152/FoxO1 were detected by immunoblotting or real-time PCR, respectively. **P < 0.01, ***P < 0.001. g pGL4.15-RNF152 plasmids were co-transfected with indicated plasmids into 293 T cells for 36 h. The luciferase activity was then measured. FoxO1, but not other family members, activated RNF152 promoter-driven luciferase activity. ***P < 0.001. h The human RNF152 promoter contains one potential binding sites for FoxO1. Point mutation was highlighted with black cross. FoxO1 was co-transfected with indicated plasmids into 293 T cells for 36 h. The luciferase activity was then measured. Overexpression of FoxO1 activated the luciferase activity driven by RNF152 WT but not RNF152 mutant. i ChIP shows enrichment of FoxO1 at the human RNF152 promoter in HepG2 cells. **P < 0.01. The human GAPDH promoter was served as a negative control. The primer pairs for Chip assay were also listed in c