Figure 4. Effect of cisplatin and pemetrexed on the induction of apoptosis in A549 cells using an annexin V-fluorescein isothiocyanate/propidium iodide (PI) apoptosis detection kit. A: A549 cells were treated with cisplatin (200 nM), and pemetrexed (100 nM) alone and in combination for 72 h and then flow cytometry was used to evaluate apoptosis. Quadrant 1 shows necrotic cells; quadrant 2 shows late apoptotic cells; quadrant 3 shows early apoptotic cells; quadrant 4 shows viable cells. B: Quantification of apoptotic cells. The results were expressed as the mean±SD from three independent experiments. Significance was determined by one-way analysis of variance followed by Dunnett’s multiple comparison test at: *p<0.05, **p<0.01 and ***p<0.001 when compared with the control. C: To evaluate the expression of cleaved poly (ADP-ribose) polymerase (PARP) (Asp 214), western blotting was performed. β-Actin served as a loading control. Representative immunoblots with quantification relative to β-actin from three independent experiments are shown.