Af6237

A RIPA lysate (Beyotime, China) was utilized to extricate total proteins, which were quantified by a BCA kit (Solarbio, China), followed by subjecting them to an SDS-PAGE gel and transferring them to PVDF membranes (GE Healthcare Life, USA). Afterwards, blocking blots were performed with 5% fat-free milk, and the process was followed by incubation with the primary antibodies against KRT17 (AF5480, Affinity, USA), E-cadherin (AF0131, Affinity), N-cadherin (AF4039, Affinity), β-catenin (AF6266, Affinity), AKT (AF6261, Affinity), p-AKT (AF0016, Affinity), mTOR (AF6308, Affinity), p-mTOR (AF3308, Affinity c-Myc (AF0358, Affinity), cyclin D1 (AF0931, Affinity), cyclin E (AF0144, Affinity), CDK2 (AF6237, Affinity), CDK4 (DF6102, Affinity), Snail (AF6032, Affinity), MMP7 (AF0218, Affinity), and GAPDH (AF7021, Affinity). Then, the membranes were encoded with a secondary antibody conjugated to HRP and finally subjected to measurement with an ECL kit (Pierce, Waltham, MA, USA).

Cells were dealt with as cell cycle and apoptosis analysis described above. Whole‐cell extracts were prepared, and then separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) before being transferred to nitrocellulose membranes (Millipore, USA). Antibodies used in this study included cyclin‐dependent kinase 1 and 2 (CDK1 and CDK2; DF6024 and AF6237, Affinity, USA), cyclinA2 (18202‐AP, Proteintech, China), cyclinB1 (28603‐1‐AP, Proteintech, China), B‐cell lymphoma‐2 (Bcl‐2; 12789‐1‐AP, Proteintech, China), Bcl‐2 associated X (Bax; 50599‐2‐Ig, Proteintech, China), vimentin (ab128507, Abcam, UK), E‐cadherin (20874‐1‐AP, Proteintech, China), N‐cadherin (22018‐1‐AP, Proteintech, China), ICAM2 (DF6772, Affinity, USA), PI3K (AF6242, Affinity, USA), p‐PI3K (AF3242, Affinity, USA), AKT (4691, CST, USA), p‐AKT (4060, CST, USA), p300 (AF5360, Affinity, USA), METTL3 (ab195352, Abcam, UK) and β‐actin (4970, CST, USA), as well as anti‐rabbit IgG horseradish peroxidase‐linked antibody (7074, CST, USA). ChemiDoc XRS+ (Bio‐Rad, USA) was adopted for capturing images.

Total protein was extracted using Radio Immunoprecipitation Assay (RIPA) buffer (P0013B, Beyotime, Jiangsu, China) containing 1 × protease inhibitor cocktail [36 (link)]. Total protein was separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (IPFL00010, Merck, MA, USA). The bands were incubated overnight with primary antibodies at 4°C. Primary antibodies are listed below: cleaved caspase 3 (AF7022, Affinity, CA, USA; 1:1000), caspase 3 (AF6311, Affinity; 1:1000), cleaved poly ADP-ribose polymerase (PARP; AF7023, Affinity; 1:1000), PARP (ab191217, Abcam, Cambridge, UK; 1:1000), cyclin D1 (AF0931, Affinity; 1:1000), cyclin dependant kinase 2 (CDK2; AF6237, Affinity; 1:1000), p27 (AF6324, Affinity; 1:1000), epithelial €-cadherin (AF0131, Affinity; 1:1000), neural (N)-cadherin (AF4039, Affinity; 1:1000), IGF1R (AF6125, Affinity; 1:1000), p-PI3K (AF3241, Affinity; 1:1000), p-AKT (ab38449, Abcam; 1:1000), p-mTOR (AF3308, Affinity; 1:1000), and GAPDH (ab9485, Abcam; 1:1000). The following day, the bands were incubated with goat anti-rabbit IgG (H + L) HRP (S0001, Affinity; 1:5000) for 1 h at room temperature. The results were visualized with ECL reagent (WBKlS0010, Merck), photographed with Gel Imager System, and analyzed using Image J software (1.8.0, National Institutes of Health, USA).