Loading buffer

Total proteins were extracted from cells and tissues by RIPA lysate (#P0013B, Beyotime) according to the instructions. Protein quantization was performed for each group according to the BCA method. Loading buffer (#MB2479, Meilunbio) was mixed with SDS-PAGE. The mixture was heated in a boiling water bath at 100°C for 5 min, and the proteins were adsorbed on PVDF membrane by gel electrophoresis. The proteins were sealed with 5% skim milk solution for 90 min at room temperature and washed with PBST for 3 times, 5 min each time. The primary antibodies used in this study include HE4 (PA5-80227, 0.3 μg/mL, Thermo Fisher), collagen I (66366-1-Ig, 1 : 2000, Proteintech), collagen III (22734-1-AP, 1 : 1000, Proteintech), MMP2 (22734-1-AP, 1 : 1000, Proteintech), MMP9 (27306-1-AP, 1 : 1000, Proteintech), PI3K (67071-1-Ig, 1 : 5000, Proteintech), p-PI3K (ab182651, 1 : 5000, Abcam), AKT (10176-2-AP, 1 : 1000, Proteintech), p-AKT (28731-1-AP, 1 : 3000, Proteintech), and GAPDH (10494-1-AP, 1 : 5000, Proteintech). Primary antibodies were incubated overnight at 4°C. Then, secondary antibodies were incubated for 90 min. ECL was color exposed, and protein bands were detected by the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA), and GAPDH served as the internal reference to detect the expression level.

The following reagents were used: DMEM, FBS, penicillin/streptomycin (Meilunbio, Dalian, China), cTnI Elisa kits (Sangon Biotech, Shanghai, China), TNF-α, IL-1β, and IL-6 Elisa kits (MyBioSource, San Diego, United States), MDA kits, SOD kits, and NADH kits (Solarbio, Beijing, China), CCK-8 kits (Beyotime Biotechnology, Shanghai, China), ROS fluorimetric kits, JC-1 fluorescent dye, protein extraction kits, and BCA kits (Meilunbio, Dalian, China). IL-1 (1:100), PINK1(1:1,000), Parkin (1:1,000), AMPK(1:1,000), Akt (1:1,000), P13K(1:1,000), Tom20 (1:1,000), β-tubulin (1:1,000), mTOR (1:1,000), and an HRP-conjugated GAPDH (1:1,000) antibody were obtained from Abclonal Biotechnology (Wuhan, China). The RIPA lysis buffer and loading buffer were purchased from Meilunbio (Dalian, China). PVDF membranes were obtained from Merck (New Jersey, United States). DMSO was purchased from Macklin (Shanghai, China), and doxorubicin, breviscapine, and dexrazoxane were purchased from Widely (Wuhan, China). The Mdivi-1 inhibitor was purchased from Selleck Chemicals (Houston, United States).