Sftpd

Western blotting analysis was carried out as described [51 (link), 58 (link), 59 (link)]. Briefly, exponentially growing imPAC2 cells were seeded in 60 mm dishes and cultured for 48 h. The supernatants were collected and dissolved in RIPA lysis buffer containing protease inhibitors, while the cells were directly lysed with RIPA lysis buffer containing protease inhibitors. The samples were subjected to SDS-PAGE and transferred to PVD membranes (Millipore). The membranes were blocked in 5% fat-free skimmed milk for 1 h, then incubated with antibodies against SftpA, SftpB, SftpD (Novus Biologicals, Shanghai, China), SftpC and β-actin (Abcam, Shanghai, China, 1:500) at 4 °C overnight, and subsequently incubated with respective secondary antibodies. The proteins of interest were visualized by using the ECL Western Blotting Substrate Kit (Abcam, Shanghai, China). β-actin (from cell lysate) was used as an internal control for both cell lysate and supernatant samples.

Immunofluorescence staining was conducted as described [30 (link), 60 (link), 61 (link)]. Briefly, subconfluent imPAC2, imPAC2 organoids, and frozen sections of imPAC2 and imPAC2-simBC implants, were fixed with paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells and sections were washed with PBS, blocked with goat serum, and incubated with primary antibodies against SftpA, SftpB, SftpD (Novus Biologicals), and/or SftpC (Abcam), followed by detection with fluorescence probes labelled secondary antibodies. Negative controls were stained without primary antibodies. Cell nuclei were counterstained with DAPI. Fluorescence images were recorded with fluorescence microscope or confocal microscopy.