Fig. 6.

BBR-induced reduction of β-catenin involves cap-dependent translation. (A) Huh7 cells were treated with DMSO (−) or increasing concentrations of BBR for 16 hours after a 2-hour pretreatment with or without 4EGI-1 (25 µM) and analyzed by Western blots. (B) Western blot analysis of extracts from HepG2 cells treated with DMSO (−) or BBR for 24 hours after a 2-hour pretreatment with or without 4EGI-1. (C) Stable Huh7 cells overexpressing either a control-shRNA (Huh7-control-shRNA) or eIF4E-BP1– and eIF4E-BP2-shRNA (Huh7-4E-BP-1+2-shRNA) were treated with DMSO (−) or BBR (+) for the indicated periods of time. Equal amounts of protein were analyzed by Western blots with the antibodies indicated. The bar graphs represent the ratio of various proteins/controls observed in the Western blots. The data represent the mean ± S.D. of four independent experiments. Statistical analysis was performed using Student’s t test and indicated as follows: *P ≤ 0.05; ***P ≤ 0.001; and ****P ≤ 0.0001. (D) Total RNA extracted from stable Huh7-control-shRNA or Huh7-4E-BP 1+2-shRNA cells treated with DMSO (−) or BBR (+) for 48 hours were analyzed by qPCR for Axin2 gene expression. The experiment was repeated twice, and data represent the mean ± S.D. of two independent PCR reactions. Significant differences were determined by t test and indicated as follows: ns, P > 0.05 and *P ≤ 0.05. (E) Huh7-control-shRNA or Huh7-4E-BP 1+2-shRNA cells treated with DMSO (−) or BBR (+) for the indicated periods of time were analyzed by Western blots. FL, full-length; ns, not significant; Tr, truncated. MW, molecular weight.