Human subject sample collection

This study was approved by the research ethics committee of the First Affiliated Hospital of Soochow University, and written informed consents were obtained from all subjects. In this study, we collected lung tissues from 10 cases of COPD with hypoxaemia and 12 controls patients in our hospital. Human lung samples were obtained from patients who underwent pneumonectomy for lung volume reduction (COPD with hypoxaemia) or lung carcinoma (controls). The explants of pulmonary artery were separated from lung samples of COPD group or control group as described in our previous study [26]. Lung tissue samples and explants of pulmonary arteries were immediately stored in liquid nitrogen. Meanwhile, all participants have been offered with the written informed consent in this recruitment.

Cell culture

Human PASMCs were kindly donated by the Institute of Respiratory Disease at the Huazhong University of Science and Technology, Wuhan, China. All cells were cultured in DMEM, supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and antibiotics (Invitrogen, Carlsbad, CA, USA) in 37℃ humidified with the condition of normoxia (21% O2, 5% CO2) or hypoxia (5% O2, 5% CO2).

Transfection

Interfering RNAs targeting DNA-PK and negative control scrambled siRNAs were designed and synthetized by GenePharma (Suzhou, China). Specific interference RNA (siRNA) sequences for DNA-PKcs were selected based on a previous publication [35]. Sequences of siRNAs are as follows: siRNA-DNA-PK sense 5′-GAUCGCACCUUACUCUGUUTTdTdT-3′; siRNA-NC sense 5′UUCUCCGAACGUGUCACGUdTdT-3′.

PASMCs were seeded into 6 well-plates at density of 2 × 105 cells/well.Then cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturers' instruction.

Cell proliferation analysis

Cell proliferation was measured by using the Cell Counting Kit-8 assay kit (CCK-8, Boster, Wuhan, China) and 5-Ethynyl-2ʹ-deoxyuridine (EdU) assays (RiboBio, Guangzhou, China). According to the manufacturers' instruction, we seeded cells into 96 well-plates with proper density. Cell viability was assessed at 24 h, 48 h and 72 h separately by CCK-8. We aslo used Edu assays kit to evaluate cell viability. Each experiment was performed in triplicate.

Cell cycle analysis

The effects of DNA damage repair on the cell cycle of PASMCs were examined by Cell Cycle Analysis Kit (Biyuntian, Shanghai, China). PASMCs at a proper density were cultured in 6-well plates in normoxia or hypoxia and with or without transfection for 48 h. Then cells were harvested and fixed in 70% methanol overnight at 4 °C.Then cells were washed with cold PBS twice and stained in propidium iodide (PI)/RNaseA mixture. After incubation in the dark for 30 min at room temperature, cells were analysed by flow cytometry.

Western blotting

Cells were lysed in ice with RIPA buffer (CST, USA) containing a protease and phosphatase inhibitor for 10 min, and then the protein concentration was measured using the BCA Protein Assay kit (Biyuntian, Shanghai, China). According to the molecular weight of protein, we used different percentages of SDS-PAGE to separate protein samples and transferred them to nitrocellulose membranes. Each membranes were incubated with respective primary antibodies at 4 °C overnight with DNA-PKcs, p-DNA-PKcs, cyclin D1 and NOR1 diluted 1:500 (Abcam, USA) and anti-β-actin diluted 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then, the membranes were washed with Tris-buffered saline with Tween-20 (TBST) for four times, and then incubated with specific HRP-conjugated secondary antibodies for 2 h at room temperature. The protein bands were visualized using an ECL kit.

Co-immunoprecipitation (Co-IP) assay

Cells were cultured in a 100 mm plate to 95–100% confluence. Then, the cells in each dish were washed twice with cold phosphate-buffered saline (PBS), collected by scraping, and lysed with 1 ml of modified RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Cell lysates were collected by centrifugation at 10,000×g at 4 °C for 30 min. Clear lysates were pre-cleared by the addition of 50 μl of protein G bead slurry and incubated at 4 °C overnight with rotation. Supernatants were transferred to a new Eppendorf tube and incubated with 1 μg of rabbit anti-DNA-PKcs antibody (Abcam, USA) with rotation overnight in a cold room; this step was followed by an additional incubation for 3–4 h with protein G beads. The beads were washed three times with RIPA buffer and then boiled in 2 × SDS protein loading buffer for 5 min. Samples (20 μl) were loaded on SDS-PAGE gels for western blot analysis.

qRT-PCR

RNAiso Plus (Takara, Osaka, Japan) was used for extracting total RNA from cell lines and tumor tissues. Reverse transcriptase M-MLV (Takara) was selected for the synthesis of cDNA. Expression values of DNA-PKcs and NOR1 were respectively normalized to the internal controls β-actin. The primer sequences for qRT-PCR of DNA-PKcs, NOR1, β-actin were as follows:

DNA-PKcs: sense 5′-AAAGACTCAAAGCCCCCTGG-3′,

anti-sense5′-GACTGTCACCCGCTCATCAA-3′;

NOR1: sense 5′-CAAGCCTTAGCCTGCCTGTCAG-3′,

anti-sense 5′-GATCTTCCTCAGTTCCACCAGTGC-3′;

β-actin: sense 5′-CACAGAGCCTCGCCTTTGC-3′,

anti-sense 5′-ACCCATGCCCACCATCACG-3′.

Quantitative RT-PCR reactions were performed using SYBR Premix ExTaq™ (Takara) on an ABI Step One Plus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA), according to the operator’s manual.

Hematoxylin and eosin staining and immunohistochemistry

In hematoxylin and eosin (H&E) assays, tissue samples were immersed in 4% paraformaldehyde for 4 h and transfered to 70% ethanol. Before dehydrated through a graded series of ethanol, biopsy material were placed in processing cassettes.Then biopsy material were embedded in paraffin wax blocks and dewaxed in xylene, rehydrated in ethanol and washed in PBS, followed by staining with hematoxylin and eosin. After staining, sections were dehydrated through increasing concentrations of ethanol and xylene. For immunohistochemistry, tissue samples were fixed in formalin and embedded in paraffin. We used an immunohistochemical detection kit to measure the expression of DNA-PKcs, α-actin and PCNA (proliferating cell nuclear antigen) protein. HPASMCs were seeded in 8-well culture plates plated with cell-climbing slices. Standard immunofluorescence of the cell climbing pieces was carried out. Then, H&E and immunohistochemical staining were observed in ten microscopic fields at × 400 magnification using a fluorescence microscope (Olympus Corporation).

Pulmonary vascular morphometry

After HE staining and immunohistochemical staining of lung tissue, pulmonary vascular morphometry were performed for pulmonary vascular remodeling. As described previously [35], the vessel wall thickness was expressed as a percentage of the external diameter [(external diameter − internal diameter)/external diameter × 100%]. The assessment was limited to medium and small arteries (≤ 500 μm diameter) with complete circumferential smooth muscle layer. At least ten vessels were measured in each section.

The muscularized vessels ratio was performed basing on the α-actin immunohistochemical staining. All these arteries were classified into three groups: nonmuscularized (25% circumference with α-actin staining), partially muscularized (25 to 75% circumference with α-actin staining) and fully muscularized (75% circumference with α-actin staining).The fully muscularized vessels were calculated and expressed as the percentage of total medium and small arteries.

Animal models

The rats used in our study were male Sprague–Dawley rats and aged 3–4 months. The rat models were housed in normoxic (21% O2) or hypoxic (10 ± 0.5% O2) conditions for 28 days in chambers with 21% normoxic conditions (room air containing 21% O2 and 78% N2) or 10% hypoxic conditions (air containing 10% O2 and 89% N2) respectively. The rats were randomly divided into normal control group, hypoxic model group, NU7026 treatment group (10 or 50 mg/kg/day), each group consisted of 6 rats. At 24 h post-hypoxia, the experiments rats were daily treated by NU7026 (10 or 50 mg/kg) for 28 days. For regression experiments, rats were housed in hypoxic conditions for 28 days in chambers and then treated with NU7026 (0 or 50 mg/kg/day) for 2 weeks. All injections were administered intraperitoneally (i.p).

Statistical analysis

All results were expressed as mean ± standard deviation (SD). For the inter-group comparisons, an unpaired two-tailed Student’s t test was applied. For the multi-group comparisons, a one-way ANOVA followed by Tukey’s test as the post hoc test was applied. P < 0.05 was considered as significant. Statistical analysis was undertaken by GraphPad Prism 5.0 (GraphPad, San Diego, CA, USA) and SPSS 17.0 software (SPSS, Chicago, IL, USA).