mTORC1/2 inhibitors potentiate mitogenic signaling in rapalog resistant HCT116 cells.

(A) Proliferation, as measured by [3H]-thymidine incorporation, of the indicated cell lines after 24 hours of the specified treatments. Values represent the mean, and error bars are the standard deviation of triplicate measurements. * Denotes P<0.05 as determined using the unpaired Student's t test. (B) Immunoblot analysis of HCT116 cell lysates after 24 hours of the indicated treatments. (C) Immunoblot analysis of lysates from HCT116 cells stably transduced with control (shScramble) or sh4EBP1 constructs. Cells underwent the indicated drug treatments for 24 hours. (D) Immunoblot analysis of HCT116 cell lysates after 24 hours of the indicated treatments with antibodies recognizing tyrosine phosphorylated proteins (left panel), proteins phosphorylated on a consensus Akt substrate sequence (center panel), or a consensus PKC substrate motif (right panel). (E) HCT116 cells were treated for the indicated time periods with 100 nM Torin or 100 nM rapamycin and analyzed by immunoblot. (F) HCT116 cells were treated for 24 hours with 10 µM U0126, 100 nM rapamycin, 100 nM Torin, or the indicated drug combinations and analyzed by immunoblot. (G) Immunoblot analysis of E2F1 promoter DNA oligonucleotide pulldowns from HCT116 cell lysates after 24 hours of the indicated treatments.