Anti claudin 4

Cells were kept in culture for 72 hours. Cells collected for flow cytometry were washed with 1x PBS, trypsinised, and resuspended in PBS-2% BSA-0.05% Tween® 20. One million (1 x 10⁶) cells were used for each labelling. The primary antibodies used were: anti-CD49f-PE (BD, Pharmigen, Franklin Lakes, NJ, USA); anti-EpCAM, anti-MUC1, anti-EGFR, anti-N-cadherin, anti-E-cadherin, anti-CD66a/c/e (Biolegend, San Diego, CA, USA); anti-EpHB4 and anti-claudin-3 (R&D Systems, Inc., MN, USA); anti-vimentin, anti-claudin-4, anti-ALDH1, anti-FGFR; secondary antibodies were Alexa488 and Alexa647 (Abcam, Cambridge, UK). Unlabelled cells and with only secondary antibody labelling were included in each independent assay and considered autofluorescent. Dead cells and debris were excluded from the analysis. Cells were analyzed in a FACs-Aria flow cytometer (BD Bioscience).

Airway epithelial cells were fixed with 4% paraformaldehyde for 20 min, and permeabilized with 0.1% Triton X-100 for 5 min. The mouse lungs were fixed in 10% formalin, embedded in paraffin and cut into 4 μm sections. Fixed cells or lung tissues were then blocked in 3% bovine serum albumin (BSA) for 1 hr, and incubated with anti-Envelope (GeneTex, #GTX636915 1:500), anti-calreticulin (Abcam, #ab22683, 1:200), anti-ZO-1 (Thermo Fisher Scientific, #61-7300, 1:50), anti-Occludin (Thermo Fisher Scientific, #71–1500, 1:100), anti-Claudin-4 (Abcam, #ab53156, 1:200), anti-phospho-JNK (Affinity, #AF3318, 1:200), anti-PDE4D (Abcam, #ab171750, 1:200), anti-phospho-SGK1 (#36–002, 1:200, Millipore), at 4 °C overnight. Then, cells or lung sections were incubated with Donkey anti-Mouse IgG (H + L), Alexa Fluor^TM 488 (Thermo Fisher Scientific, #A-21206, 1:500) or Donkey anti-Rabbit IgG (H + L), Alexa Fluor™ 568 (Thermo Fisher Scientific, #A-10042, 1:500) for 1 hr at room temperature. Finally, the cell nuclei were labeled with 4’,6-diamidino-2-phenylindole (DAPI, D9542, Sigma, USA) and visualized using confocal microscopy (TCS-SP5, Leica, Germany).

Immunohistochemical staining was performed with an Envision Kit (Dako, Kyoto, Japan) in accordance with the manufacturer’s protocol using the following antibodies: Anti-ZO-1 (dilution; 1:200, Invitrogen, Camarillo, CA, USA), anti-Occludin (dilution; 1:50, Invitrogen), anti-claudin 1 (dilution; 1:400, Invitrogen), anti-claudin 3 (dilution; 1:400, Invitrogen), and anti-claudin 4 (dilution 1:200; Abcam, Cambridge, UK). In brief, the sections were deparaffinized, rehydrated, and treated by microwave heating for 20 min in Antigen Unmasking Solution (Vector Laboratories, CA, USA) as described previously with minor modifications [25 (link)]. To quench endogenous peroxidase activity, the sections were preincubated with 0.3% H₂O₂ in methanol for 20 min at room temperature, and then incubated with the primary antibodies for 60 min at room temperature. Thereafter, the slides were washed in PBS, incubated with horseradish peroxidase-conjugated secondary antibody for 30 min, visualized using 3,3′-diaminobenzidine tetrahydrochloride with 0.05% H₂O₂ for 3 min, and then counterstained with Mayer’s hematoxylin.