Mab1378

The following antibodies were used: OCT4 (SC8629, Santa Cruz Biotechnology), NANOG (MABD24, Millipore), SOX2 (AB5603, Millipore), Integrin α6 (MAB1378, Millipore), Integrin β1 (MAB1959, Millipore), PSMD11 (NBP2-59484, Novus Biologicals; Centennial, CO) and GAPDH (2118, Cell Signaling Technology). Whole-cell lysates were prepared from cells, separated on 10% SDS-polyacrylamide gel, and transferred to polyvinylidene difluoride membranes. The membranes were incubated with 5% milk in TBST (w/v) for 1 h and then incubated with primary antibodies diluted in 5% BSA in TBST overnight at 4 °C. Blots were incubated with horseradish peroxidase-coupled secondary antibodies (Promega, Madison, WI; R&D systems, Mckinley NE, MN) for 1 h, and protein expression was detected using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific, Waltham, MA). NIH ImageJ software was used for the quantification of blotting images. Uncropped and unprocessed scans for blots are in Supplementary Fig. 11. All blots were derived from the same experimental replicate and processed in parallel.

Primary antibodies used in this study were mouse anti–acetylated α-tubulin (RRID: AB_609894; T7451; Sigma-Aldrich), mouse anti-MUC5AC (RRID: AB_2314822; MS-145-P0; Thermo Fisher Scientific), rabbit anti-MUC5B (RRID: AB_2282256; sc-20119, Santa Cruz Biotechnology), mouse anti-SCGB1A1 (MAA851Hu21; Cloud-Clone Corp.), rabbit anti-p63 (RRID:AB_2256361; 619001; BioLegend), rat anti–integrin α6 (RRID: AB_2128317; MAB1378; Millipore), mouse anti-pericentrin (RRID: AB_2160664; ab28144; Abcam), rabbit anti-TRRAP (RRID: AB_10672508; HPA038203; Sigma-Aldrich), rabbit anti-TRRAP (RRID: AB_10672042; ab73546; Abcam), and human anti-Notch2 (Danahay et al., 2015 (link)). Secondary antibodies (Alexa Fluor 488–, 568–, and 647–conjugated anti–mouse, anti–rabbit, and anti–rat antibodies; Thermo Fisher Scientific) were used in immunofluorescence (IF) and FACS. Prolong gold antifade with DAPI (P36935; Thermo Fisher Scientific) was used for nuclear staining.

100,000 cells were seeded onto a Transwell insert infected 3 h later with RSV-A2-GFP at an MOI of 0.1. Uninfected control cells were incubated with the respective volume of PBS. After 20 h cells where harvested and fixed by 2% paraformaldehyde for 10 min at RT, centrifuged for 5 min at 1,500 rcf and washed once in PBS. Fixed cells were permabilized by PBS+0.5% Triton X-100 for 15 min at RT, followed by washing once in PBS. Fixed and permabilized cells were stained for p63 (Abcam ab124762) and ITGA6 (Millipore MAB1378) at a dilution of 1∶200 for 45 min, washed once, and incubated with an Alexaflour 568 and Alexaflour 647 conjugated secondary antibody (Invitrogen) at 1∶1000 for 30 min. Flow cytometry was performed in PBS using a Fortessa cytometer.

Cells were harvested by Trypsin and washed with PBS once before fixation with 4% PFA. After blocking with block buffer (IF buffer with 1% BSA) for 30 min, cells were stained with FOXJ1 (RRID: AB_1078902; HPA005714; Sigma-Aldrich) and ITGA6 (RRID: AB_2128317; MAB1378; Millipore) antibodies for 30 min, followed by incubation with secondary antibodies (Alexa Fluor 488 anti-rat and 647 anti-rabbit). The cells were suspended in 2% FBS and analyzed by flow cytometry or cell sorting. When RNA was required to be extracted and recovered after cell sorting, RNase inhibitors (N2611; Promega) were included in all of the above buffers at a concentration of at least 1:100.

Tumors were minced, then dissociated in 0.5% collagenase in HBSS for up to 1 h, and 0.05% trypsin for up to 15 min. Cells were strained through a 70 um filter, then washed twice using FACS buffer (2% BSA /PBS), then stained with the following antibodies for 30 min at 4 °C at 1:100 dilution: anti-LYPD3 (Sino Biological 11836-R213-P), anti-Trop2/TACSTD2 (R&D systems FAB650-V), anti-LY6D (E48^{60 (link)}, courtesy of Dr. Ruud Brakenhoff), and anti-CD49f/ItgA6 (Millipore MAB1378). Propidium iodide was used at a concentration of 1:1000 as a dead cell marker. During FACS, live epithelial cells were first isolated by positive ItgA6 expression, then separated based on surface marker expression. Because LY6D was rarely expressed, both LY6D-positive and -negative cells were included in the surface marker positive (SM+) population, resulting in final expression profiles SM+ (ItgA6⁺LYPD3⁺TACSTD2⁺LY6D^+/−) vs. SM- (ItgA6⁺LYPD3^-TACSTD2^-LY6D^-). Sorted cells were then used for RNA-seq and ATAC-seq. n = 4 biological duplicates from two tumors. All samples were stained and FACS analyzed with the same parameters.