Anti snail

Whole‐cell lysates were produced using a cell lysis solution from Cell Signalling Technology (Danvers, MA). The proteins were denatured by heating in loading buffer at 100°C for 10 min. Equal amounts of protein (50 μg) from all HGEC groups were separated using 8%–10% SDS‐PAGE and subsequently transferred onto PVDF membranes. These membranes were then blocked using a protein‐free rapid‐blocking solution (Beyotime Biotechnology, China) for 1 h. After the blocking step, the membranes underwent overnight incubation with designated primary antibodies at 4°C. The antibodies utilized in this investigation included anti‐β‐actin, anti‐HIC1, and anti‐Sirt7; anti‐SDC1, anti‐H3K18ac, anti‐CD31, anti‐αSMA, anti‐Snail, anti‐ERK1/2, and anti‐phospho‐ERK1/2 (ProteinTech, Wuhan, China). After undergoing five washes, the membranes were treated by secondary antibodies at ambient temperature for an hour, followed by another five washes with PBST. The protein signals were detected using an ECL system (Beyotime Institute of Biotechnology, Shanghai, China).

Proteins were extracted in RIPA Lysis buffer (Beyotime, Shanghai, China) with protease and phosphatase inhibitors. Proteins were separated on 10% SDS-PAGE gels and then transferred onto nitrocellulose membranes (Millipore Corporation, Darmstadt, Germany). After blocking in nonfat milk, membranes were incubated overnight at 4 °C with a primary antibody and subsequently with a secondary antibody for 1 h at room temperature. The antibodies used in Western blotting were as follows: horseradish peroxidase (HRP)-conjugated anti-β-actin (1:5000 dilution, Proteintech, Wuhan, China), anti-ITGAV (1:2000 dilution, Abcam, Boston, MA, USA), anti-E-cadherin (1:2000 dilution, Proteintech, Wuhan, China), anti-Vimentin (1:2000 dilution, Proteintech, Wuhan, China), anti-Snail (1:2000 dilution, Proteintech, Wuhan, China), and HRP-conjugated goat anti-rabbit IgG (1:5000 dilution, Proteintech, Wuhan, China). The bands were visualized by an enhanced chemiluminescence detection system (Tanon, Shanghai, China), analyzed by ImageJ software and normalized to the internal control β-actin.

Total proteins were extracted by lysing cells in 6‐well plate with 60 μL RIPA buffer supplemented with protease inhibitors (#G2006, Wuhan Goodbio Technology) and phosphatase inhibitors (#G2007, Wuhan Goodbio Technology) and quantified by BCA protein assay kit (#P0011, Beyotime). One microgram of proteins was separated by 10% SDS‐PAGE and transferred to PVDF membrane (#IPVH00010, Merck Millipore). The membrane was blocked with 5% Non‐Fat Milk (#A600669, Sango Biotech) for 1 hour at room temperature and incubated with specific antibody at 4°C overnight followed by HRP‐conjugated secondary antibody incubation for 1 hour at room temperature. Images of membrane with proteins were taken with imager (Bio‐rad ChemiDoc MP, Bio‐rad). The antibodies are recorded: anti‐GAPDH (#60004‐1‐lg, Proteintech), anti‐YTHDF2 (#24744‐1‐AP, Proteintech), anti‐E‐cadherin (#20874‐1‐AP, Proteintech), anti‐N‐cadherin (#66219‐1‐AP, Proteintech), anti‐VIMENTIN (#10366‐1‐AP, Proteintech), anti‐MMP9 (#10375‐2‐AP, Proteintech), anti‐MMP2 (#10373‐2‐AP, Proteintech), anti‐SNAIL (#13099‐1‐AP, Proteintech), anti‐CDK6 (#14052‐1‐AP, Proteintech), anti‐CCND1 (#26939‐1‐AP, Proteintech), anti‐CDK4 (#11026‐1‐AP, Proteintech), anti‐KLF4 (#12173S, Cell Signaling Technology), anti‐SLUG (#C1967, Cell Signaling Technology), anti‐METTL3 (#ab195352, Abcam) and anti‐SETD7 (#ab14820, Abcam).

LUAD tissues and cells were lysed with the presence of protease inhibitors. Protein concentration was assessed using a BCA kit (Solarbio). Proteins were electrophoresed on a 10% SDS‐PAGE and transferred onto a PVDF membrane, which was blocked with 5% skim milk for 1 h at room temperature. Primary antibodies, including anti‐GAPDH (1:10000; Proteintech), anti‐ZDHHC11B (1:800, Invitrogen), anti‐Snail (1:1000, Proteintech), anti‐E‐cadherin (1:1000, Proteintech), anti‐N‐cadherin (1:1000, Proteintech), anti‐MMP9 (1:1500, Proteintech), and anti‐vimentin (1:800, Proteintech), were added to the membrane and incubated overnight at 4°C. The samples were incubated with secondary antibodies. Chemiluminescence was detected using the Tanon 4600 imaging system.

KLE and AN3CA cells with stable SLERT knockdown were lysed by RIPA buffer supplemented with protease inhibitor cocktail (Roche, Basel, CH). Protein quantification was carried out using Pierce™ BCA Protein Quantitative Kit (Invitrogen). Total protein was separated by 10% SDS-PAGE gel and transferred to PVDF membrane. After blocking with 5% skimmed milk, the membrane was incubated with primary antibodies: anti-E-cadherin (#14472, CST), anti-N-cadherin (#13116, CST), anti-Vimentin (#5741, CST), anti-Snail (#13099-1-AP, Proteintech), anti-METTL3 (#86132, CST), anti-BDNF (#47808, CST), anti-TRKB (#4603, CST), and anti-GAPDH (sc-47724, Santa Cruz Biotechnology). Lastly, the PVDF membrane was developed using SuperSignal West Pico PLUS (Invitrogen).

Tissue slides were deparaffinized and then stored in methanol containing 3% hydrogen peroxide. After the background was blocked, the slides were incubated with anti-SETD8 (dilution 1:200, ProteinTech, Wuhan, China), anti-ELK1 (dilution 1:200, ProteinTech), anti-bach1 (dilution 1:200, ProteinTech, anti-Snail (dilution 1:200, ProteinTech,), anti-vimentin (dilution 1:200, ProteinTech), anti-α-SMA (dilution 1:200, ProteinTech), and anti-CD31 (dilution 1:200, Abcam, Cambridge, UK) antibodies at 4 °C overnight. The next day, the slides were cultured with secondary antibodies at 37 °C. Finally, a DAB Detection Kit (GeneTech, Shanghai, China) was applied to stain the slides, and haematoxylin was used for counterstaining.