Muc5ac

We used fluorescence-activated cell sorting (FACS) to isolate pure populations of Tomato-positive basal cells from LV-dt/EGFP-RmiRT and LV-dt/EGFP-miRT transduced cultures. These cells were then seeded into ALI cultures and differentiated for 21 days. Cells were then dissociated with Accutase (StemCell Technologies, #07920), centrifuged at 200 RCM for 5 min, and resuspended in 1mL PBS without calcium or magnesium chloride. To evaluate EGFP expression in various cell types, cells were fixed and permeabilized using the Foxp3 Fixation/Permeabilization kit following the manufacturer’s protocol (eBiosciences/ThermoFisher #005523-00, Waltham, MA, USA). Cells were stained with the following antibodies: BSND (Abcam clone EPR14270, Cambridge, UK), MUC5AC (Novus clone 45M1, Littleton, CO, USA), acetylated alpha tubulin (Cell Signaling clone D20G3 conjugated to Alexa 647), p63 (Abcam clone EPR5701 conjugated to Alexa647). BSND and MUC5AC were stained with goat anti-rabbit and goat anti-mouse polyclonal antibodies conjugated to Alexa 647 (Invitrogen/ThermoFisher; #A-21244 and #A21235, Waltham, MA, USA). Stained cells were then run on an Attune N×T Flow Cytometer (ThermoFisher, Waltham, MA, USA) and analyzed using FlowJo version 10.7 (Ashland, OR, USA).

Trachea were excised from newborn piglets and immediately fixed in 4% paraformaldehyde (EMS) in PBS for 1 hr at room temperature. Tissues were then placed in 30% sucrose and incubated overnight at 4°C, followed by quick-freezing in OCT using a dry ice/EtOH bath and stored at −80°C. Prior to immunocytochemistry, frozen blocks of tissue were cryosectioned at 7 μm followed by permeabilization in 0.3% TX-100 (Thermo-Fisher) in PBS for 20 min, and blocked in Super-Block (Thermo-Fisher) with 5% normal goat serum (Jackson ImmunoResearch) for 1 hr, all at room temperature. Tissue sections were then incubated for 2 hr at 37°C with indicated antibodies: β-tubulin IV(1:300, Biogenex), MUC5AC (1:5000, Novus Biologicals), MUC5B (1:2000, Santa Cruz). Sections were then incubated for 1 hr with secondary antibodies goat-anti-mouse Alexa-Fluor-488 and goat anti-rabbit Alexa-Fluor-555 (1:1000, Molecular Probes/Invitrogen) and phalloidin-633 (1:300, Molecular Probes/Invitrogen). Slides were imaged on an Olympus Fluoview FV3000 confocal microscope with a Plan.ApoN 60X oil lens. Images were post-processed using the Olympus imaging software, CellSens.

Right side lung lobes were flash frozen immediately after harvest and crushed to make lysates in buffer containing 137 mM Tris HCl (pH 8.0), 130 mM NaCl, and 1% NP-40. Samples were normalized to total lung protein and used to assess the abundance of IL-6, IL-33, CCL20, Eotaxin-1 (DuoSet ELISA Kits, R&D Systems, Minneapolis, MN, USA), IL-4, IL-13 (eBioscience Kits, Thermo Fisher Scientific, Waltham, MA, USA), and MUC5AC (Novus Biologicals, Littleton, CO, USA) per manufacturer's instructions.