Foxj1

The pHAECs from healthy adult patients were kindly provided by Dr. C.U. Cotton (Case Western Reserve University), expanded, and cultured as described [34 (link),35 (link)]. Briefly, cells were plated on a layer of mitomycin C (Sigma) treated 3T3 mouse fibroblast feeder cells and grown until 70% confluency in the 1:3 mixture of Ham’s F12/DMEM media (HyClone) supplemented with 5% FBS (Sigma), 24 µg/mL adenine (Sigma), 0.4 µg/mL hydrocortisone (Sigma), 5 µg/mL Insulin (Sigma), 10 ng/mL EGF (Sigma), 8.4 ng/mL Cholera toxin, and 10 µM ROCK1 inhibitor Y-2763 (Selleck Chemical LLC). After expansion, cells (passage 3 or 4) were plated on Costar transwell inserts (Corning Inc., Corning, NY), grown until confluency, then transferred to air-liquid interface where cells were maintained in 1:1 mixture of Ham’s F12/DMEM media supplemented with 2% Ultroser G (Pall Biosepra, SA, Cergy-Sainte-Christophe, France) for 3–4 weeks until they were differentiated. Differentiation was confirmed by transepithelial resistance measurements >500 ohms, and flow cytometry staining for FoxJ1 (eBioscience) and acetylated α-tubulin (Life Technologies).

Tracheal epithelial cells were collected from the lungs of Cre-inducible reporter mice and plated on 4 μm pore transwell membranes (Corning). Cultures were grown in mTec⁺ for 2 days of proliferation then switched to mTec-serum free media for differentiation. After 21 days of differentiation, the pseudostratified cultures were infected with IAV_Cre then harvested at 1 dpi (to measure actively infected cells) and 8 dpi (to measure survivor cells after the clearance of viral infection). These cultures were then fixed with 2% PFA and antigen retrieval was performed in a citrate buffer. Samples were then stained with antibodies for active infection (HA, PY102), basal stem cells (Krt5, Biolegend, clone Poly19055, cat 905501), ciliated cells (FoxJ1, eBioscience, clone 2A5, cat 14–9965), secretory cells (SSEA-1, Biolegend, clone MC-480, cat 125611) using tdTomato as an endogenous marker of infection. Wholemount membranes were mounted in Prolong Diamond (Life Technologies) then imaged on a SP5 inverted confocal microscope (Leica) and images processed using Fiji.

The following primary antibodies were used: rabbit polyclonal antibodies against GFP (MBL, 598, IF 1:1000), RFP (MBL, PM005, IF 1:1000), Deup1 (Proteintech, 24579-1-AP, IF 1:50), Myc (Santa Cruz Biotech, sc-789, IF 1:1000), γ-Tubulin (Sigma-Aldrich, T5192, IF 1:500); mouse monoclonal antibodies against Centrin-2 (Merck Millipore, clone 20H5, 04-1624, IF 1:1000), FoxJ1 (eBioscience, 2A5, IF 1:500), GFP (Invitrogen, A11120, IF 1:1000), FLAG (Sigma-Aldrich, F1804, IF 1:1000), RFP (MBL, M208-3, IF 1:1000), acetylated tubulin (Sigma-Aldrich, T7451, IF 1:1000): rat monoclonal antibodies against ZO-1 (Santa Cruz, sc-33725, IF 1:1000). The following secondary antibodies were used: Alexa Fluor 488 goat anti-mouse IgG (H+L) (Molecular probes, A11001, IF 1:1000), Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Molecular probes, A11008, IF 1:1000), Alexa Fluor 594 goat anti-mouse IgG (H+L) (Molecular probes, A11005, IF 1:1000), Alexa Fluor 568 goat anti-rabbit IgG (H+L) (Molecular probes, A11011, IF 1:1000), Cy5 goat anti-rat IgG (H+I) (Thermo Fisher Scientific, A10525, IF 1:1000).

tdTomato transgenic mice were infected with Mal/04-Cre and collected at 14 DPI. Tracheas were then fixed and stained with Actub (Abcam, clone EPR16772, cat ab179484, 1:1000) and FoxJ1 (eBioscience, clone 2A5, cat 14–9965, 1:500). Survivor cells were identified based on tdTomato expression and cells were manually scored for the presence of FoxJ1 and ACTUB in both survivor cells and uninfected cells.

Immunofluorescent staining of either differentiating cells in ALI wells or paraffin-embedded sections of ALI wells, human bronchus from healthy nonsmokers (Donor 1: age 27, female; Donor 2: age 40, female; and Donor 3: age 27, female; catalog number HuFPT111, US Biomax, Inc., Rockville, MD, USA) or mouse trachea (C57BL/6, Normal, Female, 12 weeks) was performed as described [14 (link)]. For mouse trachea, the sections were subjected to an additional 1 h of blocking at room temperature, with 20 µg/mL Affinipure Fab fragment goat anti-mouse IgG (H + L) (catalog number 115-007-003, Jackson ImmunoResearch Laboratories, Inc. West Grove, PA, USA). The following primary antibodies were used: HEYL (5 µg/mL, catalog number H00026508-M03, Abnova, Taipei, Taiwan), SCGB1A1 (5 µg/mL, catalog number RD181022220-01, BioVendor LLC, Asheville, NC, USA), KRT5 (2 µg/mL, catalog number PA1-37974, Thermo Fisher Scientific), KRT8 (5 µg/mL, catalog number NBP2-16094, Novus Biologicals, Centennial, CO, USA), MUC5AC (1.4 µg/mL, catalog number MA5-12178, Thermo Fisher Scientific), FOXJ1 (10 µg/mL, catalog number 14-9965-82, Thermo Fisher Scientific) and acetylated tubulin (5 µg/mL, catalog number T7451, Sigma Aldrich, St. Louis, MO, USA). Imaging and quantification of the number of positive cells for each marker was performed as described previously [14 (link)].