Figure 3. Effect of eIF4F inhibition on MYC protein levels.

A. The indicated cell lines were incubated with BEZ235 (200nM, 24 h). Immunoblots of cell lysate were probed with the indicated antibodies (n=2).

B. SW480 cells were incubated with BEZ235 (200nM, 24h) and immunoblots probed for the indicated proteins (n=2).

C. m⁷GTP-CAP pull down assay was performed in SW480 cells after treatment with BEZ235 (200nM, 24h), DOX (24h), Silvestrol (25nM, 24h) or solvent control. Cell lysates were incubated with m⁷GTP beads and bound proteins immunoblotted for indicated proteins. Left panel demonstrates input of cell lysate, right panel the m⁷GTP bound protein fraction (n=2).

D. SW480 and Ls174T cells were infected with a lentivirus expressing 4E-BP1(4A) under the control of a doxycycline (DOX) inducible promoter. 4E-BP1(4A) harbors four mutations on mTOR phosphosites (T37A, T46A, S65A and T70A). Cells were incubated for 24h with DOX (1µg/ml) or ethanol as control. Protein levels were determined by immunoblotting (n=2).

E. SW480 cells expressing doxycycline-inducible 4E-BP1(4A) were incubated for 24h with DOX, BEZ235 (200nM) or the combination of both and cell lysates probed for the indicated proteins (n=2).

F. SW480 cells expressing doxycycline-inducible 4E-BP1(4A) were incubated with BEZ235 (200nM), low DOX (0.001µg/ml) or high DOX (1µg/ml) concentrations for 24h. Cell lysates were immunoblotted with the indicated antibodies (n=2).

G. SW480 cells described in panel C were incubated with DOX (1µg/ml). Left panel shows FACS analysis in response to DOX (24h) or solvent control. Error bars indicate SD of biological triplicates from one representative experiment (n=3). Right panel shows a colony assay stained with crystal violet after 5 days of DOX treatment.