MML treatment induces autophagy in A549 cells.

<p>(A) A549 cells were treated with 60 µM of MML for 24 h and then morphological images were captured by phase contrast microscope (400×, P.C.). Arrows indicate the endogenous autophagosome-like vacuoles (left) and bar graph indicates the number of vacuoles per cell. Cells were treated with MML (60 µM, 24 h) and then cells were stained with MDC and LC3 antibody, respectively. MDC (bright color, middle) images were captured by fluorescence microscope and LC3 (green, right) fluorescent images were detected by confocal microscope (bar; 10 µm). Bar graph indicates the fluorescent intensity of LC3 FITC. (B) Cells were treated with MML (60 µM) for different time periods (0–24 h) and LC3 immunofluorescence staining was performed to detect autophagosomes. Nuclei were stained with DAPI. Images were captured using confocal microscope (bar; 10 µm). (C) A549 cells were treated with various concentrations of MML (0–60 µM) for 24 h. Western blot analysis was then performed with antibodies against ATG7, Beclin-1 and LC3, respectively. GAPDH was used as loading control. Bar graph indicates densitometry analysis of LC3-II/GAPDH ratio. A549 cells were treated with different concentrations of MML (0–60 µM) for 24 h (D) or 60 µM of MML for the different time course (E) and then immunoblot analysis was carried out using antibodies against p62 and LC3, respectively. GAPDH was used as loading control. (F) A549 cells were pre-incubated with or without chloroquine (CQ, 25 µM) for 1 h, then treated with MML (60 µM) for 24 h. Western blot analysis was performed using antibodies as indicated above. GAPDH was used as loading control of Western blotting. All data were expressed as mean ± SEM of three independent experiments. *p<0.05 and **p<0.01 compared with control.