Increased Autophagy by CSE in Pulmonary Epithelial Cells.

(A) Western blot analysis of LC3 in CSE-treated HBE, SAE, and Beas-2B cells. Cells were treated with various concentrations of CSE for 24 h. Far right panel shows that bafilomycin A1 enhanced the CSE-induced LC3 expression in HBE cells. Cells were pretreated with 200 nM Bafilomycin A1 for 30 min and followed by the exposure to 30% CSE for an additional 24 h. (B) Formation of autophagic vacuoles in HBE cells treated with 1% CSE for 4 h. N indicates nuclei. AVi, immature autophagic vacuoles (autophagosomes), AVd, degradative, late autophagic vacuoles (autolysosomes). (Right) EM images corresponding to HBE cell exposed to 10% CSE for 24 h scored for number of immature autophagic vacuole (AVi) and degradative autophagic vacuoles (AVd). The data are represented as AVi and AVd per 100 µm². Data are mean scores plus standard deviation. N = 15 images representative of CSE treated cells, and N = 10 images representative of control cells, at same magnification. *P<0.05 (C) Representative images of the punctuated GFP-LC3 in Beas-2B cells treated with 5% CSE or 50 nM TSA for 24 h. White bar indicates 10 µm. (D) Increased ROS accumulation in CSE-treated Beas-2B cells. Cells were exposed to 20% CSE for 1 h. (Right) CSE-induced LC3 expression was attenuated by diphenyliodonium (DPI), Apocynin or N-acetyl-L-cysteine (NAC). Beas-2B cells were pretreated with the antioxidant at the indicated concentrations for 30 min and followed by the exposure to 30% CSE for an additional 24 h. Data represent mean+/−S.D. of three independent experiments. ** indicates P<0.01.