Fig. 3.

STAT3‐dependent modulation of 4E‐BP1 phosphorylation in HCT116 cells. In A to D, cancer cells were treated with 5 nm of siSTAT3 or 2 nm of si4EBP1 for 72 h (−, treatment with siCTRL; +, siSTAT3 or si4EBP1). (A) Western blotting was performed using equal amounts of extracts with the antibodies indicated. (B) The Renilla/firefly luciferase luminescence ratio was calculated for cap‐dependent translational activity (n = 3). (C) Cell proliferation was measured by WST‐1 assay (n = 3). (D) Cell cycle distribution was analyzed with FACS. (E–H) Cells were transfected with siSTAT3, and the cells were further transfected 24 h after siRNA transfection with a bicistronic luciferase reporter and 4E‐BP1 dominant‐active mutant vector, followed by doxycycline treatment for 48 h before harvesting cells. (E) Western blotting was performed using indicated antibodies. (F) The Renilla/firefly luciferase luminescence ratio was calculated for cap‐dependent translational activity (n = 3). (G) Counting of viable cells (n = 3). (H) Cell cycle distribution was analyzed by FACS (n = 3). Data are presented as mean ± SEM. Bars (B–D and F–H) with different letters are significantly different (P < 0.05), one‐way ANOVA. For D and H, a–c for G2/M; k–m for sub‐G1, respectively.