Fig. 2.

Experimental strategy for inhibition of mTOR signaling in bladder cancer progression at in vitro study. (A) Western blot analysis for mTOR pathway downstream protein expression in Ku-7-luc cell line treated with rapamycin. The expression of p-mTOR, and p-p70S6K, the activated form of proteins, is decreased dose-dependent manner by rapamycin concentration, but the expression of p-4E-BP1 and p-elF4E is blocked at high concentration (10 µM) of rapamycin in Ku-7-luc cell line. (B) Cell viability assay of Ku-7-luc cell line treated with rapamycin. Cell viability is inhibited at high concentration (10 µM) of rapamycin on 1, 2, and 3 days compared to control, but other concentration of rapamycin is not inhibit cell viability. (C) The transfection of siRNA against pS6K or elF4E, and rapamycin in Ku-7-luc cell line. We confirmed that the transfection of siRNA against pS6K or elF4E reduced protein expression respectively, and the dual pS6K and elF4E phosphorylation inhibited by rapamycin, simultaneously. (D) Cell viability assay of Ku-7-luc cell line treated with siRNA oligonucleotide directed against pS6K or elF4E, or high concentration of rapamycin. Cells silenced for pS6K or elF4E expression exhibit significantly reduced cell viability compared to control at 2 and 3 days, but the dual pS6K and elF4E phosphorylation inhibition by rapamycin reduced cell viability more than the transfection of siRNA against pS6K or elF4E at 3 days. ^*P < 0.05; ^†P < 0.01.