Acetylated smad4

Protein extracts from OSCC cells and clinical tissue samples were prepared in radioimmunoprecipitation assay lysis buffer supplemented with a protease inhibitor (Beyotime, Haimen, China). Protein samples were separated by 10% SDS polyacrylamide gels and then transferred to a PVDF membrane (Millipore). After blocking in 5% BSA for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies specific to β-actin (1:2000; CST), N-cadherin (1:1000; CST), E-cadherin (1:1000; CST), SIRT7 (1:1000; CST), MMP7(1:1000; CST), SMAD4 (1:1000; CST), Vimentin (1:1000; CST), and acetylated SMAD4 (1:100; Proteintech, Wuhan, China), which was then probed with a horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1000) as a secondary antibody for 2 h at room temperature. β-actin was served as the internal control for all western blots. Immunoblots were visualized and quantified using the Bio-Rad system (Bio-Rad, Hercules, CA, USA).

For Immunofluorescence staining, cells were grown onto coverslips and fixed in 4% paraformaldehyde for 10 min, and blocked with in 0.05% Triton X-100 and 3% bovine serum albumin (BSA) for 30 min. The coverslips were incubated primary antibodies (N-cadherin (1:100; Abcam, Cambridge, United Kingdom), E-cadherin (1:100; Abcam, Cambridge, United Kingdom), acetylated smad4 (1:100; Proteintech, Wuhan, China)) overnight at 4 °C, followed by fluorescence-conjugated secondary antibodies (Beyotime) for 2 h at room temperature, and nuclei were stained with DAPI (Beyotime) for 5 min.