Figure 5: Everolimus Silences E2F Target Genes Through TERT-Dependent Epigenetic Histone Modification

(A) Western blotting for MCM7 in WT and TERTtg mSMC treated with 10 μmol/l everolimus in 20% fetal bovine serum (FBS). Densitometric quantification from 3 independent experiments was normalized to GAPDH and expressed as mean fold increase ± SEM (n = 6 samples/group) relative to quiescent WT mSMC. Significance levels versus quiescent cells: *p = 0.041. Significance levels versus WT: quiescent cells, #p = 0.018; FBS + vehicle, #p = 0.046; FBS + 10 μmol/l everolimus, #p = 0.012. B and C, HEK293 cells were transfected with a TERT expression vector (TERT) or empty vector (control). Cells were serum-deprived (−) and treated with vehicle or 10 μmol/l everolimus in 20% FBS. Chromatin was harvested for chromatin immunoprecipitation assays using antibodies raised against: (B) E2F-1 (significance levels versus quiescent cells: *p = 0.045. Significance levels versus control: quiescent cells, #p < 0.001; FBS + vehicle, #p < 0.001; FBS + 10 μmol/l everolimus, #p < 0.001) or (C) acetyl-histone H3 (Lys9) (significance levels versus quiescent cells: *p = 0.048. Significance levels versus control: quiescent cells, #p = 0.034; FBS + vehicle, #p = 0.043; FBS + 10 μmol/l everolimus, #p = 0.038). Nonimmune IgG was used as a negative control value. Quantification of immunoprecipitated chromatin was performed by real-time PCR using primer pairs that cover the E2F-1 site in the proximal MCM7 promoter. Ct values were normalized to Ct values of input samples and are expressed as mean ± SEM (n = 12 samples/group) relative to quiescent cells transfected with empty vector. Abbreviations as in Figures 1 and 3.



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