Anti mettl1

Western blot assay was performed as the previous study described [48 (link)]. Total proteins were extracted using RIPA buffer (#FD011, Hangzhou Fude Biological Technology) and quantified by BCA protein assay kit (#P0011, Beyotime). In total, 1 μg of proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane (#IPVH00010, Merck Millipore). After blocked with 5% Non-Fat Milk and incubated with specific antibody at 4 °C overnight, followed by HRP-conjugated secondary antibody incubation, the membrane was imaged with imager (Biorad ChemiDoc MP, Biorad). The antibodies used in this study were listed: anti-GAPDH (#5714, Cell Signaling Technology), anti-METTL1 (#ab157097, Abcam), anti-E-cadherin (#20874-1-AP, Proteintech), anti-N-cadherin (#66219-1-AP, Proteintech), anti-VIMENTIN (#10366-1-AP, Proteintech), anti-MMP2 (#10373-2-AP, Proteintech), anti-CDK4 (#11026-1-AP, Proteintech), anti-P16 (#18769, Cell Signaling Technology), anti-ATF3 (#ab254268, Abcam).

The CR-CC and CS-CC cells were collected and the total proteins were extracted by using the RIPA lysis buffer purchased from Beyotime Biotechnology (Shanghai, China). The 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was next employed to separate the target proteins according to their molecular weight. After that, the separated proteins were transferred to the poly vinylidene difluoride (PVDF) membranes and blocked by 5% skim milk solution. The membranes were probed with the primary antibodies including anti-METTL1 (1:1000, Abcam, UK), anti-β-actin (1:2000, Abcam, UK), anti-Cyclin D1 (1:1500, Abcam, UK), anti-CDK2(1:1500, Abcam, UK), anti-p27 (1:1000, Abcam, UK), anti-cleaved Caspase 3 (1:1000, Abcam, UK), anti-S100A4 (1:1000, Abcam, UK) and anti-p53 (1:1000, Abcam, UK) for 2 hours at room temperature. The secondary antibody purchased from Abcam (1:1000) was sequentially incubated with the PVDF membranes. The protein bands were visualized by using the ECL luminescence reagent (ThermoFisher Scientific, USA) and quantified by performing the Image J software (NIH, USA).