Figure 2.

PM_2.5 induces autophagy in A549 cells. A, B. PM_2.5 increases LC3B-II expression in A549 cells. A. A549 cells were left untreated (control) or were treated with 100 μg/ml PM_2.5 for 24 h, and LC3 expression was assessed by immunofluorescence staining. DAPI staining of nuclei was performed as a background stain. A representative image is shown of the rather diffuse LC3 staining for the control cells and punctuates staining for the PM_2.5 cells (left panel). Quantification of the number of cells with LC3 puncta was performed for > 100 cells from 3 independent experiments (right panel). B. Western blot of LC3 protein expression following treatment with 100 μg/ml PM_2.5 for 0, 6, 12 and 24 h (left panel). Expression of LC3B-II was determined relative to actin and standardized to 1 in untreated cells (bottom panel). Results are representative of 3 independent experiments. C. PM_2.5 increases autophagic vacuoles in A549 cells. Autophagic vacuoles were observed by Acridine orange (AO) and monodansylcadaverine (MDC) staining in untreated cells (control) or A549 cells treated with 100 μg/ml PM_2.5 for 24 h. D. PM_2.5 induces ultrastructural features of autophagy. A549 cells were treated with 100 µg/ml of PM_2.5 for 24 h and processed for electron microscopy. Note the double membrane structure of the autophagic vacuoles. We indicate the presence of degrading autophagic vacuoles (AVds). N: Nucleus. Mit: Mitochondria. Bars 0.5 μm. Representative images are shown (right panel) and the mean ± SD of data from > 100 cells in 3 separate experiments (right panel). *p < 0.05, **p < 0.01, significant compared to controls.