Af1975

The radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) was used to extract total protein from OvCA cells. Determination of protein concentration was conducted with a BCA Protein Assay Kit (Thermo). 40 μg protein samples were loaded onto fresh sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (10%). After isolating, these bands were then electro-transferred onto a PVDF membrane (Millipore, Massachusetts, USA) and then blocked with 5% skimmed milk, followed by incubation with the following primary antibodies against EIF5A2 (ab126733, 1:5000, Abcam, Cambridge, MA, USA), proliferating cell nuclear antigen (PCNA) (AF1363, 1:1000, Beyotime), Ki67(AF1738, 1:1000, Beyotime), cyclin D1 (ab16663, 1:200, Abcam), cyclin-dependent kinases 4 (CDK4) (ab108357, 1:1000, Abcam), cleaved caspase 3 (c-caspase 3) (ab32042, 1:500, Abcam), Bax (AF1270, 1:1000, Beyotime), Bcl-2 (AF1225, 1:1000, Beyotime), (E-cadherin (ab40772, 1:10,000, Abcam), N-cadherin (ab76011, 1:1000, Abcam) and Vimentin (AF1975, 1:1000, Beyotime), or anti-GAPDH (ab181602, 1:10,000, Beyotime) at 4 °C overnight and then incubated with secondary antibodies for 1 h. The integrated optical density was quantified with Image J software (NIH, Bethesda, MD, USA) after detecting the protein bands using the ECL system (Thermo).

Cells were seeded in six-well plates and cultured for 24 h before being fixed by 4% paraformaldehyde, and incubated with specific primary antibodies vimentin (1:100; AF1975, Beyotime) and cytokeratin-7 (1:100; AF1822, Beyotime, China) at room temperature for one hour. Then, these were incubated in the dark with secondary antibodies against vimentin and cytokeratin-7. The staining was developed using a fluorescence detection system (Beyotime, China). The samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) to visualize the cell nuclei. After washing, representative images were examined under a fluorescence microscope (Leica, FL, USA).