(A) Effect of treatment with rapamycin (inhibitor of mTOR) and Bacillus Calmette-Guérin (BCG) on viability of bladder cancer cells. Cells were treated with indicated concentrations of rapamycin (1 μM, BCG, 30 MOI) for 1, 2, and 3 days. Absorbance was measured at 540 nm and cell viability was determined by the MTT assay. * p < 0.05, ** p < 0.01, ratios were normalized to each untreated group. Data are the mean ± SEM (n = 6). (B) Western blot analysis of 5637 and T24 bladder cancer cells cultured in 2D and 3D compared the expression of mTOR-mediated p70s6K and 4E-BP1 signal pathways. Cells were treated with rapamycin (1 μM) for 24 h and then the cells were lysed. Western blotting results showed a reduced level of expression in cells in the 2D environment due to treatment with rapamycin. GAPDH was used as a loading control. A representative result from three experiments is shown.