Additional file 1 of HBP1-mediated transcriptional repression of AFP inhibits hepatoma progression

Additional file 1: Figure S1. 5-FU promotes the suppression of HBP1 on AFP through enhancing HBP1 binding to AFP promoter. A-C. 5-FU promotes the suppression of HBP1 on AFP protein (A), mRNA (B) and promoter (C). HepG2 cells were transfected HBP1 with or without 5-FU treatment. The protein levels of HBP1 and AFP were measured by Western blotting. The mRNA level of AFP was measured by Realtime-PCR. The luciferase activities were detected in 293 T cells co-transfected with AFP promoter and HBP1 with or without 5-FU treatment and expressed as the means ± S.D. of the means from three experiments. (D) 5-FU promotes the interaction between HBP1 and AFP promoter. Two hundred ninety-three T cells were transfected HA-HBP1 with or without 5-FU treatment. The region from position − 1600 to position − 1448 contains the HBP1 affinity site and was analyzed by specific PCR. (E) HBP1 protein level is elevated in the presence of 5-FU. HepG2 cells were treated with different concentrations of 5-FU. The protein level was measured by Western blotting (top panel). The mRNA level was measured by Realtime-PCR (bottom panel). (F) 5-FU inhibits HBP1 ubiquitination-mediated proteasome degradation. HepG2 cells were treated with 5-FU with or without MG132. The protein level was measured by Western blotting (top panel). Two hundred ninety-three T cells were transfected HA-HBP1 with or without 5-FU treatment for 24 h and then exposed to MG132 for another 6 h prior to lysis. HBP1 protein was isolated by immunoprecipitation and analyzed by anti-Ub antibody (bottom panel). (G) 5-FU enhances the effect of HBP1 on PTEN, caspase-3, and MMP9 protein levels. HepG2 cells transfected with HBP1 were treated with or without 5-FU. The protein levels were measured by Western blotting. (H) 5-FU enhances HBP1-mediated decrease of cell proliferation. MTT assay was conducted with HepG2 cells stably transfected with control vector, HBP1 with or without 5-FU treatment. Error bars represent S.D. *, p < 0.05, **, p < 0.01.