Fig. 2
Icaritin inhibited AFP expression by increasing p53 protein expression. a Icaritin inhibits the activity of AFP promoter. Three AFP-Luc fusion constructs (pLuc1–3) with different length fragments (1871, 448 and 215 bp) were transfected into HepG2 cells following 20 μM icaritin treatment. The luciferase activities were expressed as mean ± S.E. *, p < 0.05. b Schematic diagram of the AFP promoter. Shown is the p53 affinity site within the AFP promoter at positions − 910 to − 940 and − 730 to − 760 from the translational start site (Luc-AFP, top). ChIP assay was performed to detect p53 affinity in AFP 5′ promoter region and the effect of icaritin on it. The regions from position − 702 to position − 840 and from position − 868 to − 973 were analyzed by specific PCR. Anti-p53 antibody was used in the indicated lanes. ChIP realtime PCR was performed to identify the effect of icaritin on p53 affinity. c Mut-Luc1-AFP is a mutant AFP promoter with a mutation in the p53 affinity site (bottom). Three p53-binding defective mutants (luc1–700mut, luc1-900mut or luc1–700/900mut) were transfected into HepG2 cells following icaritin treatment. The luciferase activities were expressed as mean ± S.E. d Icaritin decreased AFP expression and increased p53 expression. AFP, p53, p-p53, Arfp14 and GAPDH (as a control) protein were detected by western blot in HepG2 and SMMC7721 cells after DMSO or icaritin treatment (top). P53, AFP and GAPDH (as a control) protein were detected by western blot in HepG2 cells and SMMC7721 cells with vector, shp53, vector+icaritin or shp53 + icaritin (bottom). Results are representative of three independent experiments, and values are the mean ± S.E. *, p < 0.05; **, p < 0.01. The full-length images for gels/blots in Fig. 2b and d were presented in Supplementary Fig. 5