Fig. 4.

Snail influences mTOR/4E-BP1-controlled translation and polysome formation. a, b Snail-expressing a or -knockdown b cells and their respective controls were transfected with a bicistronic luciferase reporter that detects cap-dependent translation of the Renilla luciferase gene and cap-independent translation of the firefly luciferase gene. After 24 h, cells were treated with mTOR kinase inhibitors AZD8055 (100 nM), INK128 (100 nM), or DMSO for 12 h, followed by measurement of luciferase activities. The ratio of Renilla/firefly luciferase activities was calculated and presented as a percentage of the cap-dependent translation activity found in the DMSO-treated control cells. c Cap-dependent translation analysis of Snail wild-type (WT) and knockout (KO) HCT116 cells expressing 4E-BP1 shRNA or control shRNA treated with 100 nM INK128 for 12 h. d, e Snail-expressing T47D cells d or Snail-knockdown HCT116 cells e and their respective controls were treated with 100 nM AZD8055, 100 nM INK128, or DMSO for 12 h. Cell lysates were precipitated with m⁷GTP sepharose beads or immunoprecipitated with eIF4E antibody followed by western blot analysis for the indicated proteins. f–i Snail-expressing or vector control MCF7 cells f, g and Snail WT or KO HCT116 cells h, i were treated with 100 nM INK128 or DMSO as control for 12 h, followed by polysome analysis. The vertical blue line separates the polysomal (P) and monosomal (M) fractions. The P/M ratio, an index of translational efficiency, was calculated by comparing areas under the polysome and monosome peaks using NIH image J. The results are expressed as a percentage of the P/M ratio relative to the DMSO-treated vector g or Snail WT i controls. j, k Nascent protein synthesis was measured for Snail-expressing or vector control T47D cells j and Snail WT or KO HCT116 cells k treated with 100 nM INK128 or DMSO for 12 h. The results are expressed as a percentage of methionine analog incorporation relative to the DMSO-treated control cells. All graphic data are presented as mean ± SEM (n = 3 technical replicates per condition). *P < 0.01; ^# P < 0.001; NS, not significant using Student’s t-test