Combination treatment with pemetrexed followed by ITF2357 leads to autophagy induction in human NSCLC cell lines. (A) Flow cytometric analysis of acid vesicular organelles (AVOs) by acridine orange staining in H1299 and A549 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 48 h ITF2357). The percentage of cells with prominent red fluorescence, indicative of AVOs formation, is shown. Each panel is representative of three independent experiments with comparable results. Representative images of fluorescence microscopy in H1299 cells stably transfected with EGFP-LC3B vector (H1299/EGFP-LC3) (B), and in H1299 cells stably transfected with ptf-LC3B vector (H1299/ptf-LC3) (C) exposed to pemetrexed (0.1 μM) or ITF2357 (1 μM) alone or in combination (24 h pemetrexed followed by 48 h ITF2357). (D) Quantification of cells positive for EGFP (green) and mRFP (red) autophagosomal structures treated as indicated in B and C. The results represent the mean ± SEM of three independent experiments. (E) Western blot analysis of p62/SQTSM1, Beclin1, ATG7, LC3B-I/II protein expression in H1299 cells treated as reported in (A). HSP72/73 is shown as loading and transferring control.