Naja nigricollis toxin-γ (Sigma, St Louis, MO, USA) was purified according to the procedure previously described . N-acetylcysteine, digitonin, MTT, propidium iodide, SB202190, SP600126, U0126, rotenone, antimycin A and cyclosporin A were obtained from Sigma. Anti-p38 MAPK, anti-phospho-p38 mitogen-activated protein kinase (MAPK), anti-extracellular signal-regulated kinase (ERK) and anti-phospho-ERK, anti-c-Jun N-terminal kinases (JNK), anti-phospho-JNK, anti-caspase-9, anti-poly(ADP-ribose) polymerase (PARP), anti-Bcl-2 and anti-Bax antibodies were products of Cell Signaling (Danvers, MA, USA). Anti-caspase-3 antibodies, anti-caspase-8 antibodies and Z-VAD-fmk were purchased from Calbiochem (San Diego, CA, USA), and anti-β-actin antibodies were obtained from Chemicon (Billerica, MA, USA). Anti-cytochrome c, anti-Bak, anti-Bcl-XL and anti-Bid antibodies were products of BD Pharmingen (San Jose, CA, USA), and horseradish peroxidase-conjugated secondary antibodies were obtained from Pierce (Rockford, IL, USA). H2DCFDA and rhodamine-123 were products of Molecular Probes (Carlsbad, CA, USA). Cell culture supplies were purchased from GIBCO/Life Technologies Inc (Carlsbad, CA, USA). Unless otherwise specified, all other reagents were of analytical grade.
Cell viability assay
Human lymophoma cancer cell line U937 obtained from ATCC (Rockville, MD, USA) was grown in Roswell Park Memorial Institute medium (RPMI), 1640 medium supplemented with 10% foetal calf serum (Gibco BRL), 2mM L-glutamine, 100 U/ml penicillin/streptomycin and 1% sodium pyruvate incubating at 37°C in a humidified air containing 5% CO2. Exponentially growing cells (1 × 105) were plated in 96-well plates and treated with a series of concentrations of toxin-γ in serum-free medium after 24 hrs of growth. For pharmacological experiments, culture cells were pre-treated with 2 mM N-acetylcysteine, 10 μM SB202190, 10 μM SP600126, 10 μM U0126, 1 μM cyclosporin A, 10 μM antimycin A or 1 μM rotenone before toxin was added. At suitable time intervals, MTT solution was added to each well at a final concentration of 0.5 mg/ml and incubated for 4 hrs. Formazan crystals resulting from MTT reduction were dissolved by addition of 100 μl dimethyl sulfoxide (DMSO) per well. The absorbance was detected at 595 nm using a plate reader.
Following induction of apoptosis, cytosolic and pellet (mitochondrial) fractions were generated using a digitonin-based subcellular fractionation technique. Briefly, 1 × 107 cells were harvested by centrifugation at 800 ×g, washed in PBS and repelleted. Cells were digitonin-permeablilized for 5 min. on ice at a density of 3 × 107/ml in cytosolic extraction buffer (75 mM NaCl, 1 mM NaH2PO4, 8 mM Na2HPO4, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml aprotinin and 0.05% digitonin). Following centrifugation step 800 ×g at 4°C for 10 min., the supernatant was separated from the pellet comprising mitochondria and cellular debris. The supernatant containing cytoplasmic protein was further purified by centrifugation at 13,000 ×g at 4°C for 10 min. The pellets were solubilized in the same volume of mitochondrial lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 2 mM ethylene glycol tetraacetic acid (EGTA), 0.2% Triton X-100, 0.3% NP-40, 1 mM phenylmethylsulfonyl fluoride,5 μg/ml leupeptin, 5 μg/ml aprotinin). After centrifugation at 12,000 ×g at 4°C for 10 min., the supernatant were collected and used as the mitochondrial fraction. Cytochrome c and proteins of Bcl-2 family were detected by Western blotting analysis.
Separation of human peripheral blood mononuclear cells
Blood was obtained from three healthy adult volunteers (10 ml plus 0.1 ml of heparin, 1000 U/ml). The blood was centrifuged at 2000 rpm with a vasculant rotor for 10 min. at room temperature. The layer of white cells plus some red blood cells was taken and transferred to tubes with PBS and centrifuged at 1000 rpm for 10 min. The white layer was taken, completed to 10 ml PBS, and placed on 5 ml Ficoll-Hypaque, and after centrifugation at 2000 rpm for 30 min., the monocyte layer was taken and cultured in RPMI 1640 containing 10% foetal calf serum.
After specific treatments, cells were incubated in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 1 mM Na3VO4, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor mixtures for 20 min. on ice. After insoluble debris was precipitated by centrifugation at 13,000 ×g at 4°C for 15 min., the supernatants were collected and assayed for protein concentration using the Bradford method. An equal amount of protein per sample (15 μg) was resolved on 10% SDS-PAGE and transferred onto a polyvinyl fluoride (PVDF) membrane. The transferred membranes were blocked for 1 hr in 5% nonfat milk in PBST (PBS containing 0.05% Tween 20) and incubated with appropriate primary antibodies and horseradish peroxidase-conjugated secondary antibodies. The immune complexes were detected by SuperSignal West Pico Chemiluminescent substrate kit (Pierce) and quantified by imaging densitometry. Mean densitometry data from independent experiments were normalized to the control.