RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco-BRL (Carlsbad, CA, USA). TransFast™ Transfection Reagent was obtained from Promega Corporation (Madison, WI, USA). NF-κB luciferase reporter plasmid was a gift from Dr Luan Haojiang (US National Institutes of Health). Antibodies to cyclin D1, IκBα and p-IκBα were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Nuclear extract kit was purchased from Active Motif (Carlsbad, CA, USA). Chemiluminescent electrophoretic mobility shift assay (EMSA) kit was purchased from Beyotime Institute of Biotechnology (Nantong, China). All other reagents used in the study were of analytical grade and purchased locally.
BmK venom was extracted by mild electrical stimulation of the telsons and dissolved in 0.02 M phosphate buffer, pH 7.2, and centrifuged at 10,000 x g for 15 min at 4°C. Gel chromatography was utilized to isolate partial peptide fractions from crude scorpion venom. Seven fractions were obtained and named scorpion venom components (SVC)I, II, III, IV, V, VI, VII, respectively. The molecular weight of SVCIII was calculated to be approximately 70–80 kDa through comparison with protein markers of known molecular weights run in a 12% SDS-PAGE.
Cell culture and treatments
The THP-1 (human acute monocytic leukemia) cell line was provided by the Southern Medical University, and the Jurkat (human T lymphoma) cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were grown in 50-ml plastic flasks in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 μg/ml streptomycin, and 100 U/ml penicillin and incubated in a 5% CO2 humidity incubator at 37°C. The medium was refreshed three times a week. Cells in log phase were seeded in sterile 6-, 24- or 96-well plates with a fixed number in each well and then treated with varying amounts of SVCIII for 48 h.
Cell viability assay by MTT
Cell viability was determined by MTT assay. Cells were seeded in a 96-well plate at a density of 1x105 cells per well and treated with various concentrations (0, 1, 5, 10, 20, 30, 40 and 50 μg/ml) of SVCIII for 48 h. MTT dye was added to each well for the last 4 h of treatment. When purple precipitates were visible, the medium was carefully discarded. The formazan crystals were dissolved by adding 200 μl of dimethyl sulfoxide to each well. The cell viability index was calculated by measuring the absorbance value at 570 nm.
Flow cytometry for cell cycle analysis
A cell cycle assay was performed using propidium iodide (PI) staining of the nuclei. Following treatment for 48 h with SVCIII, cells were fixed in 70% cold alcohol overnight and then centrifuged. The pellet was re-suspended in 500 μl PI staining buffer (250 μg/ml PI, 10 μg/ml RNase in PBS) in a dark room for 30 min at room temperature and analyzed with a flow cytometer. For each measurement, at least 10,000 cells were counted.
NF-κB luciferase reporter luciferase assay
To determine the effect of SVCIII on NF-κB activation, cells were transiently transfected with a NF-κB luciferase reporter plasmid. Cells were seeded in 24-well plates (105/well) and transfected with 0.5 μg of a NF-κB luciferase reporter plasmid or pGL3 basic as a negative control using TransFast™ Transfection Reagent according to the manufacturer’s instructions and co-transfected with 40 ng of pRL-TK Renilla luciferase vector to control transfection efficiency. Transfected cells were exposed to SVCIII for 6 h. Cells were then harvested and lysed according to the manufacturer’s instructions. Supernatants were analyzed for firefly and Renilla luciferase activity using the dual-luciferase reporter assay system.
To assess NF-κB activation, EMSA was performed according to the manufacturer’s instructions for the Chemiluminescent EMSA Kit. Biotin-labeled double-stranded oligonucleotides were used which included commercially available consensus NF-κB gel shift oligonucleotide 5′-biotin-AGTTGAGGGGACTTTCCCAGG-3′. Specific binding was confirmed by competition experiments with a 100-fold excess of unlabeled or mutated oligonucleotides. The bands were detected by enhanced chemiluminescent (ECL) assay kit.
Cell extracts and western blotting
Nuclear extracts were isolated using a nuclear extract kit. Cells were briefly washed twice with ice-cold PBS/phosphatase inhibitors and incubated in 500 μl of hypotonic buffer for 15 min on ice. Subsequently, 25 μl detergent was added and the cells were vortexed at the highest setting and centrifuge suspended for 30 sec at 14,000 x g at 4°C. Nuclei were washed with 50 μl complete lysis buffer and vortexed for 10 sec at the highest setting. Thereafter the lysate was incubated for 30 min on ice and centrifuged for 10 min at 14,000 x g. Protein concentrations were determined using the Bradford assay. Proteins were resolved by 12% SDS-PAGE gels, transferred onto a PVDF membrane and subjected to western blot analysis using anti-cyclin D1, IκBα and p-IκBα antibody. Proteins were visualized with an ECL assay kit according to the manufacturer’s instructions.
Data are presented as mean ± S.D. and one-way analysis of variance was used to identify significant differences among the results. Statistical significance was defined as P<0.05.