Cyt c

HepG2 cells were fixed in 4% paraformaldehyde at room temperature for 30 min and then permeabilized using 0.05% Triton X-100 at 4 °C for 4 h [52 (link)]. Then, cells were washed with cold PBS to remove free Triton X-100. Subsequently, samples were incubated with primary antibodies at 4 °C overnight. The primary antibodies used in the present study were as follows: Cyt-c (1:500; Abcam; #ab90529), F-actin (1:500, Abcam, #ab205), p-JNK (1:500; Cell Signaling Technology, #9251) and Tom20 (1:500, Abcam, #ab186735). Tom20 was used to observe mitochondrial fission according to previous study [53 (link)]. Immunofluorescence was assessed under an Olympus IX81 microscope using FV10-ASW 1.7 software [54 (link)].

Immunohistochemical staining was conducted on 4‐mm sections of heart tissue using Mff 1:500 (Abcam), phospho‐endothelial nitric oxide synthase (p‐eNOS; Ser1177) 1:200 (Abcam), intercellular adhesion molecule–1 1:500 (Abcam), vascular cell adhesion molecule–1 1:500 (Abcam), F4/80 1:500 (Abcam), and plasma albumin 1:500 (Abcam). The primary antibodies were as follows: CD31 (1:1500, Abcam), vascular endothelial cadherin (VE‐cadherin; 1:1000, Abcam), cyt‐c (1:500, Abcam), Mff (1:1000, Abcam), pDrp1 (1:500, Abcam), HK2 (1:500, Abcam), cleaved caspase3 (CL.caspase3; 1:1000, Abcam), and pro‐caspase3 (1:2000, Abcam). 4′,6‐Diamidino‐2‐phenylindole dihydrochloride (Sigma‐Aldrich, USA), lysosome stain, and a mitochondrion‐selective MitoFluor stain (Molecular Probes, USA) were used to marker the nuclear, lysosome, and mitochondria, respectively. For the cross‐linking of VDAC1, treated cells were harvested and added with dimethyl sulfoxide as a vehicle control (2%, as used in compound‐containing samples) or cross‐linked with 0.5 mm ethylene glycolbis (succinimidyl succinate) for 10 minutes at 30°C followed by 20 mm Tris‐HCl (pH 7.4) to quench the reaction. Samples were determined by SDS‐PAGE via immunoblotting.

AEE (99.5%) was prepared in Lanzhou Institute of Husbandry and Pharmaceutical Sciences of CAAS (Lanzhou, China). MS-gradeacetonitrile was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Formic Acid (98.0%, for LC-MS) was purchased from Tokyo Chemical Industry (Shanghai, China). Catalase assay kit was purchased from Solarbio (Beijing, China). Glutathione peroxidase (GPx), GSH and GSSG assay kit, superoxide dismutase (SOD), and malondialdehyde (MDA) assay kit were purchased from Beyotime (Shanghai, China). Caspase-3 assay kit was purchased from Jianglai Chemical Biotechnology (Shanghai, China). The antibodies of Caspase-9, Caspase-3, Bax, Bcl-2, Cyt C, AIF, and IgG were purchased from abcam (Shanghai, China). Alanine aminotransferase kit and aspartate aminotransferase kit were purchased from Mlbio (Shanghai, China).

Vero E6 cells and IPI-2I cells (porcine intestinal epithelial cells) were cultured in Dulbecco’s Minimal Essential Medium (DMEM; Invitrogen) with 10% fetal bovine serum (FBS; Invitrogen) and antibiotic-antimycotic solutions (100×; Invitrogen). The cells were maintained at 37°C in a humidified 5% CO₂ incubator. The SADS-CoV was isolated from intestinal tract contents of SADS-CoV-infected piglets in Guangdong Province, China, and identified by physicochemical and neutralization testing and RT–PCR and sequence analyses [15 (link)]. SADS-CoV propagated in Vero E6 cells and virus titers was determined by 50% tissue culture infective doses (TCID₅₀) as previously described [16 (link)]. Z-VAD-FMK (R&D Systems), Z-IETD-FMK (BD Pharmingen), Z-LEHD-FMK (BD Pharmingen), and cyclosporin A (CsA; Cell Signaling Technologies) were dissolved in dimethylsulfoxide (DMSO) and stored at −20°C. The SADS-CoV N protein-specific monoclonal antibody (mAb) was prepared by our laboratory [17 ]. Antibodies specific for caspase-3, -8 and -9 were obtained from Santa Cruz Biotechnology. The PARP, GAPDH, Fas, FasL, Bid, Bax, Cyt c, apoptosis-inducing factor (AIF), and prohibitin antibodies were purchased from Abcam.

The tissues (epicenter ± 0.5 cm) were homogenized and extracted using RIPA buffer (Beyotime, Jiangsu, China) with PMSF at 3 days after SCI. After measured by the bicinchoninic acid (BCA) assay, the protein (30 μg/lane) was firstly separated through 10 or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to 0.2 or 0.45 μm polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, United States). After blocking for 1 h at room temperature with 5% skimmed milk, the membranes were incubated with the following primary antibodies against Cyt C (Abcam, United States), Bcl-2 (CST, United States), Bax (CST, United States), caspase-3 (CST, United States), cleaved caspase-3 (CST, United States), caspase-9 (CST, United States), cleaved caspase-9 (CST, United States), PARP (CST, United States), cleaved PARP (CST, United States) and β-actin (CST, United States) overnight at 4°C. After washing with tris buffered saline tween (TBST), the membranes were incubated with the goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP secondary antibodies (Abcam, United States) for 2 h at room temperature. The membranes illuminated with enhanced chemiluminescence substrate (Millipore, Billerica, MA, United States) were photographed with Image Quant LAS 4000 (GE, United States) and analyzed by Image J software (NIH, Bethesda, MD, United States).