Regulation of cell cycle distribution and ATM/pH2AX pathway by MST312. A: Western blot analysis shows an increase in pH2AX, p21 and p27 protein levels after 72 h incubation with MST312, while hTERT, dyskerin (DKC1), pAkt and pErk remain unchanged (H460 cells). B: Immunofluorescence for pATM (red) and pH2AX (green) in H1299 cells, before and after MST312 treatment. An increase in both pATM and pH2AX (which colocalize) is detected as a result of the exposure to the telomerase inhibitor; DAPI counterstaining, in blue. C: Immunoprecipitated chromatin was quantified by QPCR for the detection of telomeric sequences bound to pH2AX. Expression values were normalized with endogenous GAPDH. TRF2 binding to telomeres was used as positive control (U: Untreated, T: MST312 Treated). D: Representative immunoprecipitation PCR bands (U: Untreated, T: MST312 Treated). E: Cell cycle distribution underlines an arrest in the G2/M phase after treatment, as revealed by an increasing amount of cells in this phase and a decrease in the S phase. F: 10 days treatment with MST312 induces a dose-dependent increase in active caspase-3, H460 line. Data and error bars are presented as mean ± SEM; *: p < 0.05 compared with untreated control.