Caspase 3 and cleaved caspase 3

Cells were harvested and lysed in a strong RIPA Lysis Buffer (Beyotime) with a serine protease inhibitor phenylmethanesulphonyl fluoride (PMSF, Beyotime). Cell lysates were denatured at 100°C for 10 minutes, and protein concentrations were determined by a BCA protein assay kit (Beyotime, Taicang, Jiangsu, China). Then, 10% or 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) gel was used to separate proteins with different molecular weights, and the Trans‐Blot Turbo transfer system (Bio‐Rad, USA) was used to transfer proteins onto PVDF membranes (Millipore, USA). After blocking with 5% bovine serum albumin (BSA) at RT for about 2 hours, primary antibodies against Tubulin (1:1000; Proteintech), CDK2 (1:1000, Cell Signaling), CDK4 (1:1000; Cell Signaling), Cyclin E2 (1:1000; Cell Signaling), E‐cadherin (1:1000; Cell Signaling) and β‐catenin (1:1000; Cell Signaling), MMP2 (1:1000; Cell Signaling), Snail (1:1000, Cell Signaling), PARP and cleaved PARP (1:1000; Cell Signaling), caspase‐3 and cleaved caspase‐3 (1:1000; Cell Signaling) were incubated at 4°C overnight, and then HRP‐conjugated secondary antibodies (Life Technology) were used and incubated for 2 hours. Proteins were finally visualized by an ECL Western blot analysis system (Clinx Science) by using the ECL reagent (Beyotime).