Silencing of TS expression in H1299 cells enhances the apoptotic effect of pemetrexed and ITF2357 alone or in combination. Analysis of TS mRNA (A) and protein (B) expression evaluated respectively, by quantitative RT-PCR (A) or Western blot analysis (B) in H1299 cells exposed to pemetrexed (Pem, 0.1 μM) or ITF2357 (from 0.1 to 1 μM) alone or in combination (24 h pemetrexed followed by 24 h ITF2357). (A) Results are presented as the mean ± SEM of 2 independent experiments. p values were calculated between untreated and treated cells (*p < 0.05). (B) HSP72/73 expression was used as loading and transferring control. Western blot representative of two independent experiments with similar results is shown. Analysis of (C) viable cells evaluated by CellTitleGLO, and (D) apoptotic cells evaluated by AnnexinV/PI staining in HI299 cells transiently transfected with control RNA interference (H1299/Cont) or RNA interference directed against TS (H1299/siTS) exposed to pemetrexed (0.1 μM) or ITF2357 (1 μM) alone or in combination (24 h pemetrexed followed by 48 h ITF2357). Analysis of (E) viable cells evaluated by CellTitleGLO and (E) apoptotic cells evaluated by AnnexinV/PI staining in HI299 exposed to ITF2357 or pemetrexed as reported in C) in absence or presence of the pan-caspase inhibitor zVAD (50 μM). (C-F) p values were calculated between untreated and treated cells (*p < 0.05). The results represent the mean ± SD of three independent experiments.