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.