Figure 7: Treatment of cultured PC3 (+/−Cav1) or Cav1-deficient LNCaP malignant epithelial cells with supernatants derived from Cav1-silenced HS5 fibroblasts fostered radiation resistance. | Scientific Reports

Figure 7: Treatment of cultured PC3 (+/−Cav1) or Cav1-deficient LNCaP malignant epithelial cells with supernatants derived from Cav1-silenced HS5 fibroblasts fostered radiation resistance.

From: Progression-related loss of stromal Caveolin 1 levels fosters the growth of human PC3 xenografts and mediates radiation resistance

Figure 7

(A,B) PC3 (shCav1-transfected tumor cells [PC3(−)] as well as PC3 shCtrl control cells [PC3(+)] with normal Cav1 expression) and Cav1-deficient LNCaP cells were irradiated with 10 Gy and subsequently treated with supernatants (SN) derived from cultured Cav1-silenced HS5(−) or control transfected Cav1-expressing HS5(+) fibroblasts with or without radiation treatment (+/−XRT with 10 Gy). Western blot analysis of whole protein lysates was performed after 48 hours of treatment using the indicated antibodies. (C,D) The degree of apoptosis was quantified by measuring the SubG1 fraction 48 hours after radiation by flow cytometry analysis. Therefore, PC3cells (+/−Cav1) and Cav1-deficient LNCaP cells were left non-irradiated (white bars) or irradiated with 10 Gy (black bars) and subsequently treated with SN derived from cultured Cav1-silenced HS5(−) or Cav1-expressing HS5(+) fibroblasts with or without radiation treatment (+/−XRT with 10 Gy). (E) Quantitative Real Time RT-PCR (qRT-PCR) analysis of the reactive fibroblasts markers Acta2, Tagln as well as the tumor-promoting factors Vegf (vascular endothelial growth factor), Tgfb and Mmp2 (matrix metalloproteinase 2) were performed in total RNA isolates of Cav1-silenced HS5(−) or control transfected Cav1-expressing HS5(+) fibroblasts with or without radiation treatment (+/−XRT with 10 Gy) and were shown as relative expression to actin (set as 1) at 96 hours post irradiation. Shown are mean values ± SEM from 4 independent samples per group measured in duplicates each. *P ≤ 0.05, ***P ≤ 0.005, by one-way ANOVA followed by post-hoc Tukey’s test. (F) Expression levels of the Tgfb receptors TGFBR1 and TGFBR2 as well as the E-cadherin repressing EMT gene Snai2 were analyzed in whole RNA isolates of cultured PC3 cells (+/−Cav1) with or without radiation (48 hours after XRT with 10 Gy) using qRT-PCR analysis. Shown are mean values ± SEM from 4 independent samples per group measured in duplicates each. *P ≤ 0.05 by one-way ANOVA followed by post-hoc Tukey’s test.

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