Pro spc

ATII cells or paraffin-embedded human lung tissue sections were incubated with SP-C, TOP1-cc (both from Millipore), SP-A (Novus Biologicals, Littleton, CO, USA), pro-SP-C, p-DRP1, TOM20, MFN1, OPA1, FIS1, p63, CD68, TDP1 or DJ-1 (all from Santa Cruz Biotechnology). Secondary antibodies Alexa Fluor 594, Alexa Fluor 488 or Alexa 647 (Invitrogen Corp., Carlsbad, CA, USA) were applied for 1 h. Mitochondrial nucleoids were identified by DNA-intercalating dye Picogreen (Lumiprobe, Hunt Valley, MD, USA) as previously described [29 (link),31 (link)]. Sections were mounted with Vectashield medium containing DAPI (Abcam) to detect nuclei. Images were obtained using a confocal laser-scanning microscope (Zeiss). Pearson's correlation coefficient was used to analyze the colocalization of proteins of interest and TOM20 in SP-A-positive ATII cells in non-smokers, smokers, and patients with emphysema. Protein fluorescence intensity and colocalization were quantified by Image J (NIH) and normalized to control non-smokers as 1.

Primary AT2 cells were obtained as previously described (45 (link)). In brief, lungs were lavaged three times with phosphate-buffered saline (PBS) containing 1% antibiotics, then digested with dispase II (2 mg/ml) and deoxyribonuclease I (0.3 U/ml) at 37°C for 45 min, and minced. The digested lung pieces were filtered through 100- and 40-μm cell strainers and centrifuged. Cell pellet was resuspended with DMEM containing 10% FBS and cultured at 37°C for 45 min in CD16/32- and CD45-coated dishes to remove inflammatory cells. The supernatant was further cultured at 37°C for 45 min to remove fibroblasts. Then, cells were resuspended with DMEM/F12 containing 10% FBS, seeded in collagen I (BD)–precoated plates, and cultured at 37°C with 5% CO₂. AT2 cells were characterized by immunofluorescence staining using antibody against Prospc (Santa Cruz Biotechnology).