Fig 2: Combination of atypical PKC inhibitors and rapamycin decreases bladder cancer cell progression. (A) Proteins involved in cell cycle progression. Cells were grown in 100-mm cell culture plates, followed by treatment with either ICA-I (7.5 μM), ζ-Stat (7.5 μM), rapamycin (100 nM) or a combination. The treated lysates were then subjected to western blot analysis to observe the expression of cell cycle regulatory proteins. (B) Western blotting bands of cyclin D1 and CDK4 expression. (C) Expression levels of cyclin D1 and CDK4 presented as corresponding band intensity. (D) Western blotting bands of phospho and total p53 and MDM2, as well as p21 and p27 expression in TCCSUP cells following 3 days of treatment of ICA-I, ζ-Stat and rapamycin alone or in combination. (E) Densitometric analysis of the phospho and total p53, and MDM2, p21 and p27. on β-actin was used as a loading control (different β-actin loading controls represent the protein bands from different blots). All experiments were performed for at least three times. Data are presented as the mean ± SEM. *P<0.05, **P<0.02, ***P<0.01 and ****P<0.0001 vs. untreated control cells. MDM2, mouse double min 2 homolog; p, phosphorylated; PKC, protein kinase C.