Lc3b 2

The homogenized ventricles tissue or cultured cells were incubated in lysis buffer (pH 7.4) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 25 mM NaF, 1% Triton-X 100, 1% NP-40, 0.1 mM Na₃VO₄, 12.5 mM b-glycerophosphate, 1 mM phenylmethanesulfonylfluoride (PMSF) and complete protease inhibitor cocktail. Insoluble heart tissues or cell fractions were removed by centrifugation at 13,000 × g, 4°C for 30 min. The concentration of the extracted proteins was measured by bicinchonininc acid assay (Sigma-Aldrich, St. Louis, MO, USA). The extracted proteins were boiled for 3–5 min. in 5× loading buffer. The expression of AT1 receptor, LC3b-II, beclin-1 and phosphorylation of Akt (p-Akt) were detected by immune-blotting with antibodies against AT1 receptor (catalogue no.sc-1173G; Santa Cruz Biotechnology, Santa Cruz, CA, USA), LC3b-II (catalogue no. 2775; Cell Signaling Technology, Danvers, MA, USA), beclin-1 (catalogue no. 3495; Cell Signaling Technology), and p-Akt (catalogue no. 9275; Cell Signaling Technology), and detection was performed by using an ECL Western-blotting Detection Reagents (RPN2106; GE Healthcare, Little Chalfont, Buckinghamshire, UK) visualized densitometry with LAS-300 Image software.

Hispidulin was purchased from Aladdin Bio-Chem Technology Co., Ltd (Shanghai, China) and dissolved in dimethyl sulfoxide (DMSO) (0.1%). Bax, cleaved-caspase-3, cleaved-caspase-9, LC3B-I, LC3B-II, and β-actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). DAPI, DMSO, Dulbecco's modified Eagle medium (DMEM), and fetal bovine serum (FBS) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit and terminal deoxynucleotidyl transferase-meditated dUTP-fluorescein nick end labeling (TUNEL) in situ cell death detection kit were from Roche Diagnostics (Indianapolis, IN, USA). The bicinchoninic acid (BCA) protein assay kit was from Thermo Fisher Scientific, Waltham, MA, USA.