Snai2

Total protein was extracted from cells, lung tissues of patients, and xenograft tumour tissues of mice using RIPA lysis buffer (Elabscience). Total protein was subjected to 10% SDS-PAGE, and then transferred to polyvinylidene difluoride membranes. Polyvinylidene difluoride membranes were blocked with 5% non-fat milk in TBST for 1 h at 37 °C. After blocking, the membrane was incubated with primary antibodies (CHCHD4, cat no. 21090-1-AP, Proteintech, Wuhan, China; Bcl-2, cat no. 26593-1-AP, Proteintech; Cleaved-PARP, cat no. #5625, Cell Signalling, USA; Bax, cat no. 50599-2-Ig, Proteintech; E-cadherin, cat no. A20798, Abclonal, Wuhan, China; N-cadherin, cat. ab280375, Abcam, USA; vimentin, cat no. 10366-1-AP, Proteintech; MYC, cat no. 10828-1-AP, Proteintech, USA; Snai1, cat no. 13099-1-AP, Proteintech; Snai2, cat no. 12129-1-AP, Proteintech; and Twist1, cat no. A7314, Abclonal,; GAPDH, cat no. 10494-1-AP, Proteintech) overnight at 4 °C. Membranes were then incubated with horseradish peroxidase-conjugated goat polyclonal anti-rabbit IgG secondary antibodies (cat. no. ab150077; Abcam) for 1 h at room temperature. The target bands were visualised using a chemiluminescence kit (Beyotime).

Cells were plated onto a 48-well plate at 2 × 10⁴ cells/well and cultured for 24 h. Then, fixed with 4% paraformaldehyde for 30 min at room temperature and subsequently permeabilized with 0.5% Triton X-100 for 20 min at room temperature. The slides were then treated with goat serum for 30 min at room temperature, incubated with the following primary antibodies: Trio (1:300, Abcam), Myh9 (1:100, Proteintech), β-tubulin (1:100, Proteintech), Collagen I (1:300, Abcam), GM130 (1:100, Abcam), Pax7 (1:1000, Proteintech), Snai2 (1:1000, Proteintech), Foxd3 (1:1000, R&D Systems), Sox9 (1:1000, Cell Signaling Technology) on the slides at 4 ºC overnight. The coverslips were treated with fluorescence-conjugated secondary antibodies and phalloidin (Cytoskeleton) diluted in the blocking solution for 1 h. Then, the cells were washed with PBS and the coverslips were mounted with DAPI before imaging the cells for 90 seconds at room temperature. Photos were taken by laser confocal scanning microscopy (Carl Zeiss, Heidenheim, Germany) and Leica DM 4000 Fluorescence System.

The proteins isolated from cells using RIPA buffer and PMSF were centrifuged at 12,000 rpm for 15 min at 4 ºC, boiled for 5 min. Equal levels of proteins were separated into 10% or 12% SDS-PAGE, a 6% SDS-PAGE used to detect Trio, and then transferred into PVDF membranes, blocked with 5% non-fat milk for 2 h and then incubated with primary antibodies against the following epitopes: Trio (1:200, Abcam, USA), Myh9 (1:100, Proteintech), Rac1 (1:500, Cytoskeleton), Cdc42 (1:250, Cytoskeleton), PAX7 (1:1000, Proteintech), SNAI2 (1:1000, Proteintech), FOXD3 (1:1000, R&D Systems), SOX9 (1:1000, Cell Signaling Technology), GAPDH (1:8000, Bioworld), Histone H3 (1:1000, Cell Signaling Technology) overnight at 4 ºC. The membranes were washed with Tris buffered saline (TBST) 3 times and then incubated with secondary antibody (1:8000, KPL, USA) for 1 h at room temperature, washed with TBST 3 times again. The enhanced chemiluminescence (Thermo Fisher Scientific, Rockford, IL) was used to visualize proteins. Proteins were imaged with Chemiluminescence gel imaging system (Tonon 5200) and semi quantified by Image J software.