Cleaved caspase 3 c caspase 3

Hearts were stained with antibodies against IL-6 (goat polyclonal; Bio-techne, Minneapolis, Minnesota), CD68 (rabbit polyclonal; Abcam, Cambridge, United Kingdom), and cleaved caspase-3 (c-caspase-3) (rabbit polyclonal; Cell Signaling, Danvers, Massachusetts) with the use of species-specific secondary antibodies (Dako, Glostrup, Denmark).
c-Caspase-3 expression of nonmyocytes was measured in the area of infarction in the apical slice. Positively stained cells were counted within a grid of 100 200-μm² squares (total area: 4 mm²) and expressed as cells/mm². Further details are provided in the Supplemental Methods.

Antibodies specific for LC3B, p62, mTOR, Phospho‐mTOR, p70S6K, Akt, Phospho‐Akt, Cleaved caspase 3 (C‐caspase3), Bax, Cox‐2 and TNF‐α were purchased from Cell Signalling Technologies. Antibodies against iNOS, Bcl‐2, Iba1 and Arg‐1 were purchased from Abcam. The antibody of p‐p70S6K was obtained from Santa Cruz Biotechnology, and the antibody of CD16/32 was purchased from BD Biosciences Pharmingen. AS‐IV was purchased from MedChemExpress. Lipopolysaccharide (LPS) and tert‐butyl hydroperoxide (TBHP) were purchased from Sigma‐Aldrich. 3‐methyladenine (3‐MA) was purchased from Selleckchem. 4', 6‐diamidino‐2‐phenylindole (DAPI) was obtained from Beyotime. The polymerase chain reaction (PCR) primers were synthesized by Sangon Biotech, and other reagents used in the quantitative real‐time polymerase chain reaction (qPCR) were ordered from Takara Biomedical Technology. Horseradish peroxidase‐labelled secondary antibodies were purchased from Abcam, and AlexaFluor 594 and AlexaFluor 488 AffiniPure secondary antibodies were purchased from Yeasen.

Cells were harvested and lysed in ice-cold RIPA lysis buffer with added protease and phosphatase inhibitor (Cell Signaling Technology, Danvers, MA USA). Equal amounts of protein were resolved and separated on an SDS/PAGE gel (BioRad TrisGlycine 4–20% gel), transferred onto PVDF membranes, and subjected to immunoblot analyses. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline, pH 7.4, containing 0.05% Tween 20, and were incubated with primary and secondary antibodies according to the manufacturer’s instructions. Blotting was performed using antibodies targeting PLK1 (Abcam, Cambridge, UK; Cat. #: 17056), DNA methyltransferase 1 (DNMT1) (BD Biosciences, San Jose, CA USA; Cat. #: 612618), Bcl-xL (Cell Signaling; Cat. #: 2764), Cleaved PARP (c-PARP) (Cell Signaling; Cat. #: 9541), p53 (DO-1) (Santa Cruz, Cat. #126), p-p53 (Ser15) (Cell Signaling; Cat. #: 9284), STAT3 (BD Biosci, Cat. #: 610189), p-STAT3 (Tyr705) (abcam; Cat. #: 76315), Cleaved Caspase-7 (c-Caspase-7) (Cell Signaling; Cat. #: 9491), Cleaved Caspase-3 (c-Caspase-3) (Cell Signaling; Cat. #: 9541), and Vinculin (Cell Signaling; Cat. # 4650). Dilutions used for Cleaved Caspase-3 and Cleaved Caspase-7 were 1:250, and for other antibodies were according to the company’s recommendations.

Gambogic acid (98%) was purchased from Nanjing Jingzhu Bio-technology Ltd. (Nanjing, Jiangsu, China). Lysine was purchased from Beijing Solarbio Science and Technology Co. (Beijing, China). Gambogic acid lysinate (GAL) was made in our department. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were obtained from Sigma Aldrich (Shanghai, China). FOXO3a siRNA and the scrambled siRNA (NC siRNA) control were supplied by Santa Cruz Technology (Dallas, TX, USA). Antibodies against SIRT1, FOXO3a, p-FOXO3a (s294), p27Kip1, caspase-3 and cleaved-caspase-3 (C-caspase-3) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody against β-actin was purchased from Santa Cruz Technology (Dallas, TX, USA). Secondary antibodies were purchased from Cell Signaling Technology.