Cellview glass bottom 4 compartment dishes

To quantify autophagosomes, corrected (SFTPC^tdT/WT) or mutant (SFTPC^I73T/tdT) iAEC2s were transduced with an LC3:GFP encoding lentivirus (Twig et al., 2008 (link)) at an MOI of 20 in CK+DCI media containing polybrene (5 mg/ml). iAEC2s were then replated in growth factor reduced 3D Matrigel (Corning) at a concentration of approximately 400 cells/μl in CK+DCI media for expansion. Single cell suspension of transduced iAEC2s was achieved by incubation with 2 mg/ml dispase (Thermo Fisher Scientific) for 30–60 minutes at 37°C and subsequent incubation with 0.05% trypsin for 12–15 minutes at 37°C, as previously described (Jacob et al., 2019 (link)). Transduced iAEC2s were plated onto Greiner CELLview glass-bottom 4-compartment dishes (Greiner Bio-One GmbH) pre-coated with growth factor reduced Matrigel (Corning) in CK+DCI media. After 3 days, iAEC2s were imaged at 37°C and 5% CO₂, using a Zeiss LSM 710-Live Duo Scan confocal microscope (Carl Zeiss AG, Oberkochen) (488 nm excitation), and GFP+ punctate autophagosomes were quantified by visual inspection in 40–50 cells per group before and after 1 hour of incubation with 50 nM bafilomycin A1 (LC Laboratories). LC3 and p62 were further quantified by western blot as described below.