Top1 cc

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.

Western blotting was performed as we described previously [17 (link),19 (link),30 (link)]. Briefly, cells were lysed and lung tissue was homogenized. Mitochondrial fraction was prepared using subcellular fractionation kit (G-Biosciences, St. Louis, MO, USA) per manufacturer's instructions. Protein samples were separated by SDS-PAGE electrophoresis (Thermo-Fisher) and transferred to nitrocellulose membranes. We used the following antibodies: DRP1, TOM20, mtTFA, MFN1, MFN2, POLγ, TDP1, DJ-1 (all from Santa Cruz Biotechnology), TOP1-cc (Millipore), p-DRP1 (Ser616) (Cell Signaling Technology, Danvers, MA, USA), GAPDH (Abcam, Cambridge, MA, USA) and β-actin (Sigma, St. Louis, MO, USA). We used horseradish peroxidase (HRP)-conjugated AffiniPure donkey anti-rabbit immunoglobulin (Ig) G or anti-mouse IgG purchased from Jackson ImmunoResearch (West Grove, PA, USA). The blots were developed using an enhanced chemiluminesence kit for Western blotting (Millipore) according to the manufacturer's instructions. Images were quantitated using NIH Image J software.