M199 and DMEM media, fetal bovine serum (FBS), antibiotics (penicillin and streptomycin), and other cell culture supplements were obtained from Invitrogen (Carlsbad, CA). Protease and phosphatase inhibitor cocktails were obtained from Roche Molecular Systems, Inc. (Alameda, CA). Molecular weight markers and reagents for protein determination were from Bio-Rad (Cambridge, MA). Antibodies against E2F1, CCNE1, β-actin, and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G were from Santa Cruz Biotechnology (Santa Cruz, CA). Polyvinylidene difluoride (PVDF) membrane was obtained from Amersham Pharmacia Biotech. Other reagents used in this study were obtained from Sigma (St. Louis, MO.) otherwise as stated.
Isolation and culture of chicken pulmonary arterial smooth muscle cells (PASMCs)
All animal care and procedures were in accordance with institutional and international guidelines. Twenty day-old chicken embryo pulmonary arterial smooth muscle cells (PASMCs) were isolated by enzymatic digestion as previously described[19, 20]. In brief, proximal pulmonary arteries were isolated under aseptic condition and were cut into small pieces after removal of fat, adventitia, and connective tissue surrounding the arteries. After incubation for 90 min in DMEM medium supplemented with elastase type III (0.125 mg/mL), collagenase type I (0.5 mg/mL), and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin), the tissues suspension was centrifuged for 15 min at 1,500 g and the pellet was resuspended in M199 media supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 8 mmol/L HEPES, and 2 mmol/L glutamine at 39°C in a humidified atmosphere of 5% CO2/95% air environment. PASMCs were passaged with 0.05% trypsin and cells of passages 3–8 were used in all the experiments. 95% percent cells exhibited specific immunostaining by anti-α-smooth muscle-actin antibody, a generally accepted marker of smooth muscle cells as expected.
Cell proliferation assay
Proliferation of PASMCs was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay as previously described. Briefly, cells were seeded in 96-well plates at a density of 2,000 cells each well under normoxic or hypoxic conditions for indicated time points. At the end of treatment, 10 μL MTT (5 mg/mL)/well was added to each well of the plates, and incubated for another 4 h at 37°C. The supernatant was then carefully removed, and 75 μL/well dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance of the solubilized product at 570 nm was measured with microplate spectrophotometer (PowerWave XS, BioTek Inc, Vermont).
[3H] Thymidine incorporation assay
Thymidine incorporation assay was performed according to the method described previously[20, 22]. Briefly, PASMCs were seeded in complete growth medium at a density of 20,000 cells/well in 24-well plates. After 24 h, cells were growth-arrested by serum starvation with low serum medium (0.1% FBS) for 48 h. Arrested cells were labeled with [3H]-thymidine at 25Ci/mL and then were subjected to normoxia or hypoxia. Cells were harvested at indicated time points in 1% sodium dodecyl sulfate (SDS)/0.01 mol/L NaOH. [3H] thymidine incorporation was determined in a Becton scintillation counter (model LS6500, Franklin Lakes, NJ).
Cell Cycle Analysis
Cell cycle was determined by flow cytometry as described previously. Treated cells were harvested and washed two times with cold PBS, and then were fixed with 1 mL of 70% ethanol overnight at 4°C. Fixed cells were centrifuged for 3 min at 1,200 g and then were washed with cold PBS and resuspended in PBS with 50 mg/mL prepodium iodide (PI) and 1 mg/mL RNase. The stained cells were analyzed for DNA content by fluorescence-activated cell sorting (FACs) in a FACs Calibur (Becton Dickinson Instrument, San Jose, CA). Cell cycle fractions were quantified using the CellQuest software (Becton Dickinson).
Quantitative real-time PCR
Total RNA was extracted from the PASMCs using an RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer's instructions and estimated using optical density measurements. Total RNA (2 μg) was used for first-strand cDNA synthesis using random hexamer primers and Superscript Reverse Transcriptase (Invitrogen, Carlsbad, CA). Quantitative real-time PCR was used to quantify the expression level of E2F1. PCR reactions were set up with SYBR Green PCR Supermix (Bio-Rad, Hercules, CA) using 1 μL of cDNA in each 20-μL reaction mixture). The relative values of gene expression were calculated using the ΔΔCT method with β-actin as the internal control. The values were normalized to the average value of the control samples.
Transfection reagent Lipofectamine-2000 was obtained from Invitrogen (Carlsbad, CA). The procedure for siRNA transfection was performed as previously described. 48 h after siRNA transfection, cells were seeded in 6-well plates and subjected to normoxia or hypoxia for another 24 h; cells were then collected for Western blot analysis or DNA synthesis assay.
Western blot analysis
The total lysate was obtained from PASMCs using ice-cold lysis buffer containing 20 mmol/L Tris–HCl (pH 7.4), 2.5 mmol/L EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 200 mmol/L NaF, 100 mmol/L Na3VO4 and 1 mmol/L protease inhibitor cocktail from Roche (Alameda, CA). Cell lysates were centrifuged for 15 min at 16,000 g to remove cellular debris. Equal amounts of protein were separated on SDS-page gels and transfer to PVDF membranes (Millpore, Bedford, MA). The membranes were blocked in 5% non-fat milk at room temperature for 1 h, and then were incubated with primary antibodies against β-actin, E2F1, CCNE1, overnight at 4°C and then washed three times with PBST. The membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h. The signal detection was performed by using the enhanced chemiluminescence substrates (ECL Plus reagent; Amersham Pharmacia Biotech, San Diego, CA).
Comparisons between groups were performed using two-way ANOVA followed by the Duncans test. Differences were considered statistically significant at the level of P <0.05 and values are represented as means ± SEM. The statistical analysis was performed with the software of SPSS 11.0 for Windows.