Snail1

Whole-cell lysates were obtained by RIPA lysis buffer. Primary antibodies against GAPDH (1 : 1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), E-cadherin, Snail1, ZEB1, ZO-1, PI3K, AKT, phospho-AKT (1:1000, Cell Signaling Technology), and WDR5 (1:500, R&D) were used. Enhanced chemiluminescence (Perkin Elmer, Hopkinton, MA, USA) was used to visualize the protein bands transferred to the PVDF membranes. Detailed standard western blotting was performed as described previously.^{46 (link)}

To determine protein expression, we harvested cellular proteins for western blotting as previously described [20 (link)]. Cells were washed three times with ice-cold 1× PBS and lysed on ice in a cell lysis buffer (50 mM Tris-HCl, pH 8.0; 120 mM NaCl2; 0.5% NP-40; 100 mM sodium fluoride; and 200 M sodium orthovanadate) supplemented with protease inhibitors. Proteins (50 μg per lane) were separated by gel electrophoresis on 10% SDS-PAGE gels, transferred to nitrocellulose membrane by electroblotting. The membranes were probed with appropriate primary antibodies against α9-nAChR (Thermo Fisher Scientific, Rockford, IL, USA, #PA5-46826), PD-L1 (Genetex, Irvine, CA, USA, #GTX104763; Thermo Fisher Scientific, #14-5983-82; Santa Cruz Biotechnology, Santa Cruz, CA, USA, #sc-293425), total-AKT (Genetex, #GTX121937), phospho-AKT (Cell Signaling Technology, Beverly, MA, USA, #9271), total-ERK1/2 (Genetex, #GTX113094), phospho-ERK1/2 (Santa Cruz Biotechnology, #sc-7383), total-STAT3 (Santa Cruz Biotechnology, #sc-8059), phospho-STAT3 (Cell Signaling Technology, #9145), Snail-1 (Cell signaling Technology, #6615), Twist-1 (Santa Cruz Biotechnology, #sc-81417) and GAPDH (Genetex, #GTX627408) followed by incubation with an HRP-conjugated secondary antibody. Band intensity was quantified using ImageJ software. The relative protein levels were normalized against the GAPDH protein levels.

Polyclonal antibodies to Notch1, Notch2, Notch3, Notch4, Dll1, Dll3, Dll4, Jagged1, Jagged2, HIF‐1α, N‐cadherin, E‐cadherin and Snail1 were purchased from Cell Signaling Technology (Beverly, MA). Polyclonal antibodies to G6PD, transketolase (TKT), Nrf2, Keap1 and monoclonal antibody to β‐actin were purchased from Abcam (Cambridge, UK). The dual‐luciferase reporter assay kit was obtained from Promega (Madison, WI).

After coinfection with TGF-β1 and LASP1 shRNA or TGF-β1 and LASP1 pcDNA for 48 h, the whole-cell fractions and nuclear fractions from the two lung cancer cell lines were prepared using the nuclear protein extraction kit (Beyotime, Shanghai, China) following the manufacturer's instruction. The total protein contents were measured by Coomassie brilliant blue assay. The total proteins binding with SDS were electrophoresed on 10% SDS-PAGE and transferred to PVDF membranes. The membranes were immune-blotted with the primary antibody overnight at 4°C and subsequently immune-blotted with secondary antibody (Boster) for 2 h at room temperature. Blots were visualized using the ECL method (Boster). Primary antibodies of LASP1 (#8636, 1 : 1000), N-cadherin (#4061, 1 : 1000), E-cadherin (#3195, 1 : 1000), vimentin (#5741, 1 : 1000), pSmad2 (#3108, 1 : 1000), Smad2 (#3122, 1 : 1000), pSmad3 (#9520, 1 : 500), Smad3 (#9523, 1 : 500), Snail1 (#3879, 1 : 1000), GAPDH (#2118, 1 : 1000), α-tubulin (#2144, 1 : 1000), and Lamin B (#13435, 1 : 500) were purchased from Cell Signaling Technology (Massachusetts, USA). Smad7 primary antibody (#25840-1-AP, 1 : 500) was purchased from Proteintech (Illinois, USA).

We detected protein levels by western blot analysis as described previously^{81 (link)}. Cell lysates were collected into 150 µl 2× Laemmli buffer with 1 M DTT at 24, 48, or 72 h after initial exposure to stimuli (vehicle/sham, TGF-β1 at 10 ng/ml, or compression at 30 cm H₂O). The following antibodies and dilutions were used, with primary antibody diluted in 5% skim milk or 5% BSA according to the manufacturer’s instructions: E-cadherin (1:10,000), N-cadherin (1:1000), Snail1 (1:1000), vimentin (1:1000), GAPDH (1:5000), all from Cell Signaling Technology; EDA-fibronectin (1:1000, Sigma). We report fold-changes of normalized protein levels compared either to vehicle control (for E-cadherin) or to TGF-β1—treated at 72 h (for mesenchymal markers) across n = 3 donors.
We detected mRNA expression as previously described^{84 (link)}. Cells were collected from the conditions and donors as described above at 3, 24, or 48 h after the initial exposure to stimuli, and RNA was isolated from cell lysates using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. Real-time qRT-PCR was performed using primers listed in Supplementary Table 2, and fold-changes were calculated by the comparative ΔΔCt method^{150 (link)}.