Fig. 4. PES1 regulates telomerase activity and telomere length.

(A) Direct telomerase assay in PES1 KO MCF7 cells or MEFs transfected with Flag-hTERT, hTR, and PES1 or its mutants. Transfected cells were immunoprecipitated with anti-Flag, followed by telomerase activity detection. Parental cells without Flag-hTERT transfection were used as a negative control. Values shown are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01. (B) PES1 WT and KO mouse hepatocytes were isolated from liver-specific PES1 KO mice generated by mating conditional PES1 KO mice with Alb-Cre transgenic mice and were analyzed by TRAP assay. (C) IP-TRAP analysis of PES1 KO MCF7 cells transfected with Flag-tagged WT or mutant PES1. When the binding of Flag-tagged proteins to anti-Flag agarose beads was complete, the reaction mixtures were centrifuged and the supernatants were collected for TRAP assay. The precipitates were washed, lysed, and used for TRAP assay. Flag-tagged PES1 or endogenous PES1 levels were analyzed by immunoblot with anti-PES1. (D) IP-TRAP analysis of siRNA-mediated PES1 KD HepG2 cells transfected with Flag-PES1 or empty vector. (E) TRAP assay was performed by mixing in vitro transcribed hTR and in vitro translated hTERT with purified WT and mutated His-PES1. (F) Representative MCF7 monoclones stably expressing PES1 shRNA or control shRNA and WT or mutant PES1 were harvested at indicated PDLs. Reexpression of WT or mutant PES1 was performed after 25 PDLs by splitting the same clone. Telomere lengths were measured using terminal restriction fragment (TRF) analysis. Representative immunoblot reveals PES1 expression. PES1-R, shRNA-resistant PES1. PES1 W397R-R, shRNA-resistant PES1 W397R. (G) PES1 WT and KO mouse hepatocytes isolated from liver-specific PES1 KO mice at 3 months were subjected to TRF analysis. Left and right panels indicate two nest mice. fl, flox.