Fig. 6.

Added effect of STAT3 and MLST8 knockdown on cap‐dependent translation in HCT116 cells. siSTAT3 (5 nm) were transfected into cells for 48 h. The cells were then transfected with siMLST8 (1 nm) for 24 h. (A) Western blotting was performed using equal amounts of extracts with the antibodies indicated (top), and the band intensity of phospho‐4E‐BP1(S65; bottom) was quantified (n = 3). (B) siSTAT3‐transfected cells were further transfected with siMLST8 and bicistronic luciferase reporter to measure luciferase activities. Cap‐dependent translational activity of each group was compared with siCTRL group (n = 3). (C) m⁷GTP pull‐down assay was performed after serial treatment with siSTAT3 and siMLST8 (top), and the cap‐binding 4E‐BP1 index was determined by the ratio of 4E‐BP1 to eIF4E (bottom; n = 3). (D) Counting of cell numbers (n = 3). (E) Cell cycle distribution was analyzed by FACS (n = 3). Data are presented as mean ± SEM. Different letters (A–E) are significantly different (P < 0.05), one‐way ANOVA. For E, a–d for G2/M; k–m for sub‐G1, respectively.