Bm3919

Immunoblotting was performed as described previously (17 (link)–19 (link)). Protein was extracted from SUM159 cells using 1× RIPA buffer (P0013B, Beyotime, Shanghai, China). Subsequently, the proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electro-transferred onto PVDF membranes, which were incubated with primary antibodies overnight at 4°C. The protein bands were detected using enhanced chemiluminescence substrate (P0018M, BeyoECL Plus, Beyotime, China). The antibodies used were as follows: mouse anti-human E-cadherin (1:500, BF0219, Affinit); rabbit anti-human SNAI1 (1:500, 13099-1-AP, Proteintech); rabbit anti-human SMAD3 (1:500, BM3919, BOSTER); rabbit anti-human p-Smad3 (1:500, AP0328, Bioworld); rabbit anti-human N-Cadherin (1:500, No.48495-2), rabbit anti-human METTL3 (1:500, No.31591), and rabbit anti-human GAPDH (1:3000, MB001) all from Bioworld Technology, Ltd).

The cells were grafted to 2.5 × 10^4/well seed containing slides in 24-well plates and cultured for 48 h. After collecting the cells, they were fixed with 4% paraformaldehyde. Cells were permeabilized with 0.2% tritonX-100 for 1h and then blocked with 5% BSA and sequentially incubated with the primary antibodies of interest and the next day after incubation using phasic secondary antibodies, The samples were washed with saline-sodium citrate (SSC) twice, stained with DAPI, and the slides were removed and sealed with an anti-fluorescence quenched tablet, and tested by fluorescence test under laser scanning confocal microscope.
The primary antibodies used were as follows: Smad2 (A00090-1, Boster, 1:200 dilution; China); Smad3 (BM3919, Boster, 1:200 dilution; China); α-SMA (BM0002, Boster, 1:200 dilution; China); TGF-β1 (MA00019, Boster, 1:200 dilution; China).