Western blot (WB)

Total protein in tissue or cell was extracted by RIPA lysate solution containing PMSF (P0013C, Beyotime, Shanghai, China). The prepared cells were incubated on ice for 30 min, and then the supernatant was centrifuged (4 ℃, 8000 g) for 10 min. A BCA test kit was used to detect the total protein concentration (P0012, Beyotime). Fifty µg protein samples were dissolved in 2 × SDS sample buffer and boiled for 10 min, and SDS-PAGE gel electrophoresis was performed followed by transfer of proteins to a PVDF membrane. After blocking in 5 % non-fat milk for 1 h, PVDF membranes were incubated overnight with primary antibodies: METTL3 (1:1000, ab19552, Abcam, Cambridge, UK), HMGA2 (1:1000, ab207301, Abcam), E-cadherin (1:500, ab15148, Abcam), Snail (1:500, ab82846, Abcam), Vimentin (1:2000, ab137321, Abcam), N-cadherin (1:1000, ab18203, Abcam), and GAPDH (ab19485, 1:2500, Abcam). Then, PVDF membranes were incubated for 1 h with corresponding HRP labeled goat-anti-rabbit IgG H&L (HRP) (ab97051, 1:2000, Abcam). According to the instructions of the ECL detection kit (product No. BB-3501, Amersham, UK), equal amounts of liquid A and B were mixed in the dark and then dripped onto the PVDF membranes. The film was photographed using a specific image analysis system (Bio-Rad company, USA), and analyzed using Quantity-One (v4.6.2) software. The relative protein content was expressed by the gray value of corresponding protein bands/the gray value of internal reference bands. The mean of triplicate experiment results was calculated [20, 21].