Annexin 5 pi kit

Prior to analysis of ROS by flow cytometry, cells were collected after treatment which was described in Section 2.1 and incubated with a fluorescent probe 10 μM 2′,7′-dichlorofluorescein diacetate (DCFH-DA, KeyGen, Nanjing, China). Then, samples were centrifuged at 600 ×g for 5 min, washed three times with cold PBS, and analyzed by flow cytometry (excitation: 488 nm; emission: 525 nm). Cardiomyocyte apoptosis was determined by Annexin V/PI kit (BestBio, Shanghai, China). In brief, cells were washed with ice-cold PBS and collected. Then, cells were resuspended in 1x Annexin V Binding Buffer (250 μl). Cells were mixed with 5 μl Annexin V-FITC dye and incubated at 4°C for 15 min in the dark. Cells were mixed with 10 μl propidium iodide dye, incubated at 4°C for 5 min in the dark, and analyzed by flow cytometry.

Emodin was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China; Purity ≥98% by HPLC; lot number: T17O11F127680), which was dissolved in DMSO to provide a stock 50 mM solution and stored at − 20 °C. Fetal bovine serum, culture medium, and other solutions used for cell culture, where specially noted, were from Gibco (Brazil). Chloroquine (CQ), an autophagy inhibitor, was purchased from Selleck Chemicals (TX, USA). PI3K agonist 740 Y-P were purchased from MedChemExpress (Guangzhou, China). Matrigel was purchased from Corning (NY, USA). Dimethyl-sulfoxide was obtained from Sigma-Aldrich Chemical (MO, USA). EdU detection kit and ad-mCherry-GFP-LC3 were purchased from RiboBio Co., Ltd. (Guangzhou, China). Annexin V/PI kit and cell cycle detection kit were purchased from BestBio (Shanghai, China). Trizol was obtained from Invitrogen (CA, USA). The primary antibodies against E-cadherin, N-cadherin, vimentin, PI3K, p-PI3K, AKT and p-AKT were bought from Abcam (Cambridge, MA, USA). The primary antibodies against β-catenin, GSK3β, p-GSK3β, Snail, Slug, Beclin1, LC3B, SQSTM1, and p-mTOR were purchased from Cell Signal Biotechnology (Beverly, MA, USA). HRP-conjugated Affinipure Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG were sourced from Proteintech (Chicago, IL, USA).