Fig. 5: FGF2 upregulates ONECUT2 expression through the FGFR1/ERK/ELK1 signaling pathway.

A The levels of ONECUT2 were detected by RT-PCR and western blotting after treatment with different concentrations of FGF2 (0, 5, 10, 20 ng/ml) for 24 h. B Luciferase reporter assay showing the promoter activity of ONECUT2 in PLC/PRF/5 cells and SNU398 cells after FGF2 treatment (20 ng/ml, 24 h). C The levels of ONECUT2 were detected in PLC/PRF/5 cells after FGF2 treatment (20 ng/ml, 24 h) and FGFR1, FGFR2, FGFR3, or FGFR4 knockdown by western blotting. D The relative luciferase activity was detected in PLC/PRF/5 cells after transfection with truncations and mutated ONECUT2 promoter constructs, followed by FGF2 treatment (20 ng/ml, 24 h). E HCC cells were transfected with ELK1-silencing lentivirus, followed by FGF2 treatment (20 ng/ml, 24 h). Luciferase reporter assay showing ONECUT2 promoter activity. F RT-PCR and western blotting showing ONECUT2 levels in HCC cells transfected with ELK1-silencing lentivirus followed by FGF2 treatment (20 ng/ml, 24 h). G Protein levels of ONECUT2, ERK, p-ERK, JNK, p-JNK, P38, p-P38, AKT, p-AKT, PKCα, and p-PKCα were measured by western blotting when PLC/PRF/5 cells were pretreated with inhibitors of ERK (SCH772984, 10 μM, 30 min), JNK (SP600125, 20 μM, 1 h), P38 (SB203580, 20 μM, 1 h), PI3K (LY294002, 20 μM, 1 h) or PKC (GO6983, 10 μM, 30 min), followed by administration of FGF2 (20 ng/ml, 24 h). H Relative binding of ELK1 to ONECUT2 promoter was determined by ChIP assays when HCC cells were treated with FGF2 (20 ng/ml, 24 h) and the indicated inhibitor. Data are mean ± SD. *P < 0.05, **P < 0.01.