Caspase 3 antibody

β-actin, ZO-1, caspase-3 antibodies were purchased from Cell Signaling Technology (Danvers, Massachusetts). VE-cadherin antibody was obtained from Santa Cruz Biotechnology (Dallas, Texas). Recombinant Human TNF-α was from R&D Systems (Minneapolis, Minnesota). Tubastatin A, CAY10603 were obtained from Selleck Chemicals (Houston, Teaxs). Lipopolysaccharide (LPS) from Escherichia coli 0111:B4 was purchased from Sigma Aldrich (St. Louis, Missouri). Endothelial growth medium (EBM-2) was obtained from Lonza (Allendale, NJ). In Vitro Vascular Permeability Assay (96-Well) kit was purchased from EMD Millipore (Billerica, Massachusetts). Lipofectamine RNAiMAX reagent, HDAC6 siRNA and control siRNA were obtained from Life technologies (Carlsbad, California).

The AFM1 powder used in this experiment was provided by Pribolab (Qingdao, China). Dissolve AFM1 in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA) to prepare 200 μg/mL stock solution, and then dilute it to the required concentration with culture medium. Lf powder was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dissolve Lf power in culture medium to make 10 mg/mL stock solution, and then dilute it to the required concentration. Nonessential amino acids, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and Opti-MEM Medium were purchased from Gibco (Carlsbad, CA, USA). Trypsin (2.5%), antibiotics (100 units/mL penicillin, 100 μg/mL streptomycin), Nonessential amino acids (NEAA), Enhanced Cell Counting Kit-8 (CCK-8), and Annexin-V-FITC Staining Kit were provided by Beyotime Biotechnology (Shanghai, China). Trizol Reagent Kit and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA, USA). The Prime Script RT Reagent Kit (Perfect Real Time) and the TB Green Premix Ex Taq II (Tli RNaseH Plus) were obtained from Takara (Shiga Prefecture, Japan). The β-actin and caspase9 antibodies were provided by Bioss (Beijing, China); and the Atg5, SQSTM1/p62, LC3B, caspase3 antibodies, and Atg5-specific siRNAs were provided by Cell Signaling Technology (Danvers, MA, USA).

A Bruker AVANCE-500 spectrometer was used to detect NMR spectra. All chemical shifts were given relative to tetramethylsilane (TMS). Bruker 7.0 T SolariX XR FT-ICR-MS was used to record mass spectra. TLC-analysis was performed on glass-backed plates (Sigma-Aldrich, Canada) coated with 0.2 mm silica 60F254. Commercial common reagent-grade chemicals were used without further purification. The gastric adenocarcinoma cell line SGC-7901, cervical cancer cell line HeLa, lung carcinoma cell line A549, human hepatocellular carcinoma cell line BEL-7402, and normal live cell line LO2 were purchased from the cell bank of the Cell Institute of Sinica Academia Shanghai (Shanghai, China). Buffers were prepared using doubly distilled water. The 4′,6-diamidino-2′-phenylindole (DAPI), cell cycle and apoptosis analysis kits were purchased from Beyotime (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) was obtained from Sigma–Aldrich. The fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA) and 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) were purchased from Roche Diagnostics (Indianapolis, IN, USA). Polyclonal antibodies against Bcl-2, Bax, and P38 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase-3 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

The kidneys of different groups were processed by dehydration in alcohol and embedded in paraffin. Renal sections were cut at a thickness of approximately 5 μm, stained with hematoxylin and eosin (HE), and analyzed under a microscope. Images were captured with a 20× objective lens. For immunofluorescence staining, sections were incubated with caspase-3 antibody (1:100; Cell Signaling Technology, USA) at 4 ℃ overnight and then with fluorescently labeled secondary antibodies at room temperature for 2 hours in the dark. Finally, DAPI was added to the samples to stain the nuclei. Protein was observed under a laser confocal microscope. For immunohistochemistry staining, sections were incubated with KIM-1 (1:100; Absin, Shanghai, China) or NGAL (1:100; Affinity Biosciences, USA) antibody, followed by incubation with biotinylated goat anti-rabbit IgG and the ABC complex. Photographs were taken under a microscope with a 20× objective lens.