Isolation of mouse PASMCs by magnetic separation
Healthy adult C57BL/6J mice weighing about 20 g were obtained from the Laboratory Animal Center, Shanxi Medical University. After anesthesia by intraperitoneal injection of 4% chloral hydrate (0.01 ml/g), the mice were planned to expose the kidneys, and their renal arteries were severed. After the thorax was exposed, 3-5 ml of sterilized PBS solution was slowly infused from the right ventricle to the pulmonary artery until the lungs turned white. Slowly injected 3-5 ml PA agarose into the right lung until the lungs turned gray. Removed heart and lungs and placed in ice-cold PBS for about 5 min to allow the gel to coagulate. The lung tissue was shredded and then transferred to a 50 ml sterile centrifuge tube. The magnet was attached, and the ferrous tissue moved to the wall of the centrifuge tube near the magnet. The PBS solution was then aspirated, and the lung tissue was washed 3 times with 5 ml sterile PBS solution. 6 ml of collagenase solution was added to the centrifuge tube, poured into a petri dish and digested for 1 hour at 37℃. One hour later, lung tissue and collagenase were repeatedly aspirated and transferred to a sterilized centrifuge tube. The magnet was connected, and the supernatant was aspirated and washed with complete medium to inactivate the collagenase. Finally, added complete medium and then transferred the cell suspension into Petri dish and incubate overnight (5% CO2, 37℃). On the second day, the tissue pieces in the culture dish were poured into a 50 ml centrifuge tube. Connected the magnet, used a complete culture medium to clean the centrifuge tube for 3 times, and then transferred into a new sterile Petri dish, placed in 5% CO2, 37 ℃ incubator to continue culturing. 3-5 days to replace the medium. During the culturing process, magnetic separation techniques may again be used to separate the tissue fragments in the culture vessel according to cell growth. About 50% of the cells that first crawled out of the iron-containing vessels were smooth muscle cells and the other 50% were fibroblasts. Because the remarkable growth superiority of fibroblast, the culture dish was discarded to promise the better growth of smooth muscle cells. When the cells were fused to about 80%, 500 μL trypsin was added into T25 culture bottle, and the cells were left for 3-5 min at room temperature. When the cells became round, complete culture medium were added to neutralize the pancreatin, and then a pipette was used to suck the cells to promote shedding. In the first passage, after the cell suspension was made and the magnets were connected, the iron particles were seen to gather on the wall of the centrifuge tube near the magnet. The cells were collected into the T25 culture bottle with the discard of the centrifuge tube, and undergo repeated magnetic separation, the iron particles were eventually all be discarded. The cells were cultured to the desired number and state through several passages.
Immunofluorescence staining
PASMCs were fixed with 4% paraformaldehyde for 10 min, and were incubated with 0.2% permeable Triton X-100 for 15 min. Being washed three times with PBS, the cells were blocked with goat serum for 1 h, and then incubated with anti-smooth muscle (1:100) and anti-smooth muscle heavy chain (1:80) primary antibodies overnight at 4°C. Cells were then washed three times with PBS, followed by incubation with fluorescein isothiocyanate-conjugated secondary antibody and tetramethylrhodamine-conjugated secondary antibody for 1 h in the dark. After three washes with PBS, the cell nuclei were stained with DAPI for 10 min and observed by confocal laser scanning microscope. All the above procedures were carried out at room temperature.
Hypoxia treatment of mouse PASMCs
According to different hypoxia treatment times, the cells were divided into 24 h group, 48 h group and 72 h group, and each group was set up with a normoxia control group. The cells of the normoxia group were cultured in a CO2 incubator with the following culture conditions: 37℃, 5% CO2, 21% O2; the cells of the hypoxia group were cultured in a tri-gas incubator with the following culture conditions: 37℃, 5% CO2, 3% O2, 92% N2. Cell proliferation in each group was detected by using CCK-8 and Brdu method. Similarly, PASMCs with good growth status were selected and randomly divided into 0 h group, 24 h group, 48 h group and 72 h group according to different hypoxia treatment time. Western-blot and flow cytometry were used to determine the expression of PKCδ, PKCε, PKCη and PKCθ under different hypoxic induction time. In order to investigate the relationship between hypoxia-induced up-regulation of nPKCs and the proliferation of PASMCs and its possible mechanism, PASMCs were divided into the following groups according to different interventions. PKCδ: Normoxia group, Normoxia+PMA group, Hypoxia group, Hypoxia+Rottlerin group. PKCε: Normoxia group, Normoxia+PMA group, Hypoxia group, Hypoxia+PKCε inhibitor peptide group.
Small interfering RNA treatment of mouse PASMCs
For siRNA treatment, PASMCs with a growth density of approximately 70-80% of the culture flask bottom were selected. A lentiviral vector containing siRNA fragments for inhibiting PKCδ and PKCε, respectively, was constructed and the control group was set up with no-load lentiviral vector. The volume of lentiviral vector required was calculated based on MOI=40 and the concentration of the different vectors. After calculation, the lentiviral vectors were then mixed with incomplete medium and incubated in a hypoxic incubator for 72h. Group by different interventions: Normal group, Scramble group, PKCδ knockdown group, PKCε knockdown group. After treatments, western blot was used to detect the expression of PKCδ, PKCε, P-AKT, AKT, P-ERK and ERK in each group. Cell proliferation was detected by using Brdu method.
Brdu incorporation
Cells were washed three times with PBS and Brdu solution was diluted 2000 times. 5 μl of Brdu solution was then mixed with 10 mL of FBS-DMEM solution at a concentration of 10%, avoiding light throughout the process, the total volume of the mixture depends on the number of samples. Then 5 mL of the mixture was added to each flask of cells, followed by incubation in a normoxia incubator for 72 h. After the treatment, discarded the mixture and washed with PBS three times. Cells were then fixed at room temperature with 4% paraformaldehyde for 20 min. After fixation, cells were washed three times with PBS and 2 M HCl was added, followed by a 37°C water bath for 10 min. 0.1% TritonX-100 was used to break the cell membrane structure for 5 min. Being washed three times with PBS, the cells were blocked with goat serum for 1 h, and then incubated with Brdu primary antibody overnight at 4°C. Cells were then washed with PBS, followed by incubation with fluorescein isothiocyanate-conjugated secondary antibody at 37°C for 3 h. After three washes with PBS, the cells were stained with DAPI for 10 min and observed by confocal laser scanning microscope.
CCK-8 assay
CCK-8 assay was used to detect PASMCs proliferation. PASMCs were seeded in 96-wells plates at the concentration of 4×103 cells/well, then the 96-wells plates were put into normoxic and hypoxic incubators respectively to detect cell proliferation in the normoxic group for 24 h, 48 h and 72 h and the hypoxic group for 24 h, 48 h and 72 h. Each well was added with 10 μl of CCK-8 solution and continued to incubate for 2 h. Finally, the optical density value of absorbance at 450 nm was measured.
Western blotting
After treatments, cells were washed in cold PBS for three times, collected and scraped with cell lysis buffer and then kept in a 4°C refrigerator for 2 h. The lysates were then centrifuged at 12000 r/min for 15 min, the supernatants were collected, and the total protein concentrations were calculated. Proteins with equal amounts (20 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 5% BSA at room temperature for 1 h, the membranes were then incubated with antibodies specific for rabbit anti-PKCδ (1:5,000), rabbit anti- PKCε (1:1,500), rabbit anti- PKCη (1:100), rabbit anti-PKCθ (1:5,000) and rabbit anti-β-actin (1:5,000) overnight at 4°C. Subsequently, the membranes were incubated with a secondary antibody for 2 h at 4°C.
Flow cytometry
Cells in good condition were digested with trypsin free of EDTA, then washed with PBS, centrifuged at 1000 r/min for 5 min, and the cell sediment was collected. Cells were then fixed with 1% paraformaldehyde for 10 min and 0.05% Triton was used to break the cell membrane structure for 1 min. After three washes with PBS, the cells were blocked with goat serum for 1 h. Being washed three times with PBS, the cells incubated with primary antibody at room temperature for 1 h. Cells were then washed with PBS, followed by incubation with secondary antibody at room temperature for 30 min. After the treatment, washed with PBS three times and detected the result.
Statistical analysis
All statistical analyses were performed using SPSS16.0, and the results were presented as Mean ± SD. The one sample t test was used for comparison between groups. P<0.05 was considered statistically significant. The statistical graphs were drawn using Prism Graph Pad 8.01.