Isolation and culture of rat PASMCs
Twenty male Sprague-Dawley rats (age, 50 days; weight, 80-100 g) purchased from the Experimental Animal Center of Shanxi Medical University (Taiyuan, China) were maintained in a temperature(25±2˚C) and humidity ( 60 - 65%) controlled room on a 12 h light/dark cycle with free access to food and water for 1 week prior to use. The current study was reviewed and approved by the Animal Management Guidelines of the Ministry of Health of the People's Republic of China, in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rat PASMCs were isolated and cultured as previously reported. Rats were anaesthetized by intraperitoneal injection of pentobarbital sodium (Sinopharm Chemical Reagent Co., Ltd., Beijing, China; 50 mg/kg body weight), then the main trunk of pulmonary arteries and the right and left branches were isolated under a dissecting light microscope (Olympus Corporation, Tokyo, Japan). After connective tissues of arteries were cleaned and vessels cut open longitudinally, luminal endothelia were removed by gentle scraping with cotton swabs. The isolated pulmonary arteries were dissected into small pieces of 1x1 mm, maintained in Dulbecco's Modified Eagle's Medium (DMEM)/F12 media (Thermo Scientific Hyclone, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Carlsbad, CA, USA) and 1% SM cell growth supplement (SMCGS) (Science Research Laboratories, Carlsbad, CA, USA) and incubated in a humidified atmosphere with 5% CO2 at 37˚C. Culture medium was changed twice per week and cells were harvested with trypsin (0.25%; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing EDTA. Morphological assay and immunocytochemistry were used to detect SM-actin expression to identify PASMCs, Cells passage between three and six times were used in the experiments.
Treatment of rat PASMCs
All experiments were carried out as follows. The cells were seeded at a density of 5 × 104 cells/well in 6-well plates or at 1.5 × 103 cells/well in 96-well plates and cultured in DMEM/F12 media supplemented with 10% FBS and 1% SMCGS, in 5% CO2 at 37°C for 24 h. The cells were then starved in serum-free media for 24 h, so that they subsided into the resting stage. Thereafter, the media were changed to DMEM/F12 supplemented with 2% FBS and 1% SMCGS, and the cells were cultured at 37°C under hypoxic conditions (5% O2, 5% CO2, and 90% N2) for 24 h, 48 h or 72 h. Cells in the control group were cultured under normal conditions (21% O2, 5% CO2, and 74% N2).
For specific inhibition of the ERK1/2 and p38 signaling pathways, cells in the resting stage were treated with the ERK1/2-specific inhibitor U0126 (5 mM; Cell Signaling Technology, Boston, MA, USA) or the p38-specific inhibitor SB203580 (20 mM; Sigma-Aldrich, Louis, MO, USA) in DMEM/F12 supplemented with 5% FBS and 1% SMCGS and the cells were cultured at 37°C under hypoxic conditions.
KLF4 overexpression and knockdown
Cells were seeded in 6-well plates. The plasmid pCMV.SPORT6 were used, which contains KLF4 coding sequences and was obtained from the Hematology Department of the Second Affiliated Hospital, Shanxi Medical University. For KLF4 overexpression, KLF4 was cloned from this plasmid into the eukaryotic expression vector pIRES2-ZsGreen1 and transfected into resting-stage PASMCs with the Attractene Transfection Reagent (Qiagen, Dusseldorf, Germany) according to the manufacturer’s instructions. The pIRES2-ZsGreen1 plasmid was used as a negative control. After culture under hypoxic conditions for 72 h, cells were collected for further study.
For KLF4 knockdown, FlexiTube GeneSolution GS9314 for KLF4 (Qiagen, Dusseldord, Germany) or non-specific siRNA as a negative control was transfected into resting-stage PASMCs with the HiPerFect Transfection Reagent (Qiagen, Dusseldord, Germany) according to the manufacturer’s instructions. After culture under hypoxic conditions for 72 h, cells were collected for further study.
Western blot analysis
Cells were collected and subjected to lysis with lysis buffer (Beyotime, Shanghai, China) supplemented with 1 mM phenylmethylsulfonyl fluoride. Lysis buffer (50 μl) was used for the cells in each well of a 6-well plate. Bicinchoninic acid assay (Beyotime, Shanghai, China) was used for protein quantification. The proteins (50 μg) were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto nitrocellulose (NC) membranes (GE Healthcare, Buckinghamshire, UK). The NC membranes were blocked in phosphate-buffered saline with Tween 20 (PBST) containing 5% nonfat milk powder at room temperature for 1 h and incubated overnight with primary antibody at 4°C. Primary antibodies were diluted with PBST as follows: 1:200 mouse anti-human proliferating cell nuclear antigen (PCNA) antibody (Boster, Wuhan, China); 1:400 rabbit anti-human smooth muscle (SM) α-actin antibody (Abcam, Cambridge, MA, USA); 1:200 rabbit anti-human KLF4 antibody (Abcam, Cambridge, UK); 1:200 rabbit anti-human ERK1/2 antibody (Cell Signaling Technology, Boston, MA, USA); and 1:1000 rabbit anti-human β-actin antibody (Boster, Wuhan, China). Next, the NC membrane was incubated with horseradish peroxidase–labeled secondary antibody (Boster, Wuhan, China) diluted with PBST at a ratio of 1:10,000 at room temperature for 1 h, and subjected to development with electrochemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA). Signal intensity was quantified and analyzed with GeneSnap (Syngene, Cambridge, UK).
SPSS 21.0 (SPSS, Chicago, IL, USA) was used for statistical analysis. Data are presented in the form of Mean ± SD. Test of normality and homogeneity of variance was carried out. We used the t-test and one-way analysis of variance for comparisons between two groups and among three of more groups, respectively. The Student-Newman-Keuls method was used when variance was homogeneous, while Dunnetts’s T3 method was used when variance was not homogeneous. Differences were considered significant at P<0.05.