Figure 5: Reduction of Cav1 levels increased survival of clonogenic epithelial PC3 and stromal HS5 cells while proliferation was decreased in vitro.

(A) Lentiviral expression of a Cav1-specific siRNA (shCav1) in stromal HS5 fibroblasts resulted in an efficient and sustained down-regulation of Cav1 expression compared to control-transduced (shCtrl) cells as shown by Western blot analysis. β-actin (bActin) was included as loading control. Representative blots of at least three different experiments are shown. (A) HS5 (shCav1-transfected fibroblasts [HS5(−)] as well as shCtrl control cells [HS5(+)] with normal Cav1 expression) cells were plated for colony formation assay, irradiated with indicated doses (0–8 Gy) and subsequently further incubated for additional 10 days. Data show the surviving fractions from three independent experiments measured in triplicates each (means ± SD). ****P ≤ 0.001 by two-tailed students T-test. (B) Cell proliferation was analyzed by cell counting in cultured shCav1-transfected HS5(−) and control-transfected HS5(+) fibroblasts cells at the indicated time points after irradiation with 10 Gy. Data are shown as means ± SEM of three independent experiments. (C) Expression levels of the indicated proteins were analyzed in whole protein lysates of cultured HS5 cells (+/−Cav1) with or without radiation (48 hours after XRT with 10 Gy) using Western blot analysis. Representative blots are shown. For quantification blots were analyzed by densitometry and the respective signal was related to beta-actin (n = 4–5 for each group). For determination of the Akt phosphorylation status the obtained phospho-specific signal was related to the signal of the total protein (phAkt/Akt). P-values were indicated: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005 ****P ≤ 0.001; by one-way ANOVA followed by post-hoc Tukey test.