Fig. 4

Combination of curcumin and gefitinib significantly potentiates induction of autophagy and apoptosis. a H157 and H1299 cells were treated with gefitinib, or curcumin alone, or the two combination at indicated concentration for 48 h. Then the LC3-II puncta formation was determined using immunofluorescence analysis and imaged by confocal microscopy (left). The number of LC3-II puncta/cell was quantified by Image-Pro Plus 5.1 software (right). (**P<0.01 compared with gefitinib, or curcumin alone, or the two combination plus 3-MA, respectively). b H157 and H1299 cells were stained with acridine orange after drug treatments (5 μM of gefitinib or 10 μM of curcumin alone, or the two combination for 48 h) and analyzed by confocal fluorescent microscopy. Development of AVOs (orange) was observed (left). Quantification of AVOs using FACSanalysis (right) (** P<0.01 compared with gefitinib, or curcumin alone, or the two combination plus 3-MA, respectively). c H157 and H1299 cells were treated with gefitinib, or curcumin alone, or gefitinib plus curcumin at indicated concentration for 48 h. The autophagy-related and apoptosis activation-related proteins were analyzed by immunoblotting. d H157 and H1299 cells were treated with gefitinib plus curcumin in the absence and presence of Baf A1 or 3-MA. The autophagy-related proteins were analyzed by immunoblotting. e H157 and H1299 cells were treated with gefitinib, or curcumin alone, or gefitinib plus curcumin at indicated concentration for 48 h and then were fixed with methanol, immunostained with anti-EGFR (green), anti-LC3 (red), and DAPI (blue), and analyzed by confocal microscopy to determine the intracellular locations of EGFR and LC3. f H157 cells were treated with 5 μM gefitinib (Gef) plus 10 μM curcumin (Cur) for 48 h in the presence or absence of 3-MA or Beclin-1 knokdown. The phosphorylated and total EGFR were detected by immunoblotting. Similar results were obtained from three independent experiments. Typical immunoblots were presented in the Figure