Combination treatment with pemetrexed followed by ITF2357 leads to apoptosis induction in human NSCLC cell lines. Flow cytometric analysis of (A) cell cycle distribution by PI staining, and (B) apoptotic cells by AnnexinV/PI staining in A549 and H1299 cell lines exposed to pemetrexed (Pem, 0.1 μM) or ITF2357 (from 0.1 to 1 μM) given alone or in combination (24 h pemetrexed followed by 48 h ITF2357). (A) The percentage of cells in sub-G1 and in the different cell cycle phases is shown. Where not reported, the percentage of cells in sub-G1 phase was less than 10%. Each panel is representative of three independent experiments with comparable results. (B) The percentage of AnnexinV+/PI- (early apoptotic cells, lower right), AnnexinV+/PI + (late apoptotic cells, upper right), AnnexinV-/PI- (viable cells, lower left) and AnnexinV-/PI + (necrotic cells, upper left), cells is shown.