Calpain9

The cells of different groups were evenly spread on cover slips in a six-well plate and cultured to confluence. The cells were washed with PBST, fixed with methanol at −20°C for 20 min, and then wash with PBST and block with 5% BSA for 1 h and then combined with α-SMA (Proteintech, United States), cytokeratin 18 (Proteintech, United States), Vimentin (Proteintech, United States) and calpain9 (Proteintech, United States) primary antibody overnight at 4°C then washed with PBST and then incubated with fluorescent secondary antibody at room temperature for 1 h. The nuclei were counterstained with diamidinophenylindole (DAPI) (Beijing Zhongshan Jinqiao, China), and the images were observed and collected with a fluorescent inverted microscope (OLYMPUS, Japan).

Protein from cell lysates or tissues lysates were separated by SDS–polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and incubated with primary antibodies, including: calpain-9 (1:1000; Proteintech, USA), calpain-8 (1:1000; Abcam, USA), cyclin D1 (1:500; Cell Signaling, USA), cyclin D3 (1:1000; Cell Signaling), CDK4 (1:1000; Cell Signaling), CDK6 (1:1000; Cell Signaling), p21 (1:1000; Cell Signaling), cleaved caspase-3 (1:500; Cell Signaling), cleaved caspase-8 (1:1000; Cell Signaling), cleaved caspase-9 (1:1000; Cell Signaling), and caspase-12 (1:1000; Proteintech), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2000; Santa). Protein expression was visualized by enhanced chemiluminescence assay.