Epcam clone 9c4

Human lung single-cell isolation and flow cytometry analysis were performed as described previously (10 (link), 13 (link)). In brief, human lung tissues were minced and then digested with 2 mg/mL Dispase II, followed by digestion with 10 U/mL elastase and 100 U/mL DNase I. Finally, cells were filtered through a 100 μm cell strainer and lysed with red blood cell lysis to get single-cell suspensions. Antibody staining was similar in human and mouse cells. Flow cytometry was performed with a Fortessa and FACSAria III flow cytometer and analyzed with FlowJo 10.6.1 software (BD Biosciences). Anti–human CD31 (clone WM59, catalog 303118, RRID AB_2247932), CD45 (clone WI30, catalog 304016, RRID AB_314404), and EpCAM (clone 9C4, catalog 324212, RRID AB_756086) were from BioLegend. SLC39A8 (ZIP8) polyclonal antibody (catalog PA5-26368, RRID AB_2543868) and goat anti–mouse IgG/IgM (catalog A-10680, RRID AB_2534062) were from Thermo Fisher Scientific. HTII-280 (52 (link)) was a gift from the laboratory of L. Dobbs (UCSF). Human SIRT1 monoclonal mouse IgG1 (clone 834918, catalog IC7714S) from R&D Systems was used for intracellular staining. Pirfenidone was from MilliporeSigma (catalog P2116), and nintedanib was from Selleck Chemicals (catalog S1010).

Only cells without fluorescent protein tags were stained. Cells were fixed with ice-cold methanol/acetone (50/50 v/v) for 10 minutes at room temperature. The cells were incubated at 4°C overnight with the following primary antibodies: keratin 14 (RB-9020, 1:4,000), keratin 18 (MS-142, 1:2,000), keratin 19 (MS-198 1:1,000), β-casein (MS-935 1:1,000), Ki67 (RB-1510, 1:500) (Thermo Scientific, Stehelin), p63 (MS-1081, 1:200) (Thermo Scientific), e-cadherin (610181, 36/Ecad, 1:1,000) (BD Bioscience,), EpCAM (Clone 9C4, 1:100) (BioLegend), or keratin 8/18 (GP11, 1:500) (Fitzgerald). Goat anti-mouse, goat anti-rabbit or goat anti-guinea pig secondary antibodies coupled to Alexa 488, 568 or 633 (Molecular Probes, Invitrogen 1:500) were used for detection.