Adult male C57BL/6 J mice (8 weeks), obtained from the Laboratory Animal Center at the Huazhong University of Science and Technology were used in this study. All animal protocols were approved by the Institutional Animal Care and Use Committee of Ethics of Tongji Medical College, Huazhong University of Science and Technology.
Murine hindlimb ischemia model
As described previously , male c57Bl mice (8 weeks) were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight). After skin preparation, the superficial skin was incised to expose the femoral artery. The femoral artery was separated from the accompanying vein and nerve. The proximal and distal ends of the femoral artery were ligated, and the middle segment was cut to induce hindlimb ischemia.
Detection of perfusion recovery
Blood perfusion before and after surgery was monitored with a laser Doppler system (Perimed, Sweden). In detail, the mice were anaesthetized by intraperitoneal injection of pentobarbital sodium, and then the hindquarters were imaged by laser Doppler imaging. The reduction in and recovery of blood perfusion were obtained by calculating the ratio of perfusion in ischemic to nonischemic limbs.
Human umbilical vein endothelial cells (HUVEC) were isolated from fresh umbilical cords as described previously . The cells were cultured in Endothelial Cell Medium (ECM) (ScienCell) at 37 °C in a humidified incubator (Thermo Scientific, 3111) with 5% CO2. Hypoxia exposure was applied by culturing the cells in an incubator (Thermo Scientific, 3131) with 1% O2 and 5% CO2. For endothelial infection, empty adenoviral vector (Ad-Vector) and adenoviruses vector encoding KPNA2 (Ad-KPNA2), scramble short hairpin RNA (Ad-NC), and KPNA2 shRNA (Ad-shKPNA2) were applied to infect HUVEC in vitro. Viral fluid volume = number of cells × multiplicity of infection (MOI) /viral titer. Cells were lysed 48 h after infection.
293 T cells with STR identification were obtained from the American Type Culture Collection. They were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco) with 10% FBS (Gibco) and 1% volume of 100 × penicillin/streptomycin solution (Beyotime Biotechnology, C0222). The final concentration of penicillin is 100 U/ml and the final concentration of streptomycin is 0.1 mg/ml. The cells were cultured at 37 °C in a humidified incubator with 5% CO2.
Western blotting was performed as described previously . Tissue or cells were lysed using RIPA lysis buffer (Beyotime Biotechnology, P0013C) supplemented with PMSF (Thermo Scientific, 36978), protease inhibitors (Roche, 04693132001) and phosphatase inhibitors (Roche, 04906837001). Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Scientific, cat No. 23225). Western blotting was performed with antibodies against the following: KPNA2 (1:1000, Abclonal, A5012), KPNA1 (1:1000, Abclonal, A1742), KPNA3 (1:1000, Abclonal, A8347), KPNA4 (1:1000, Abclonal, A2026), KPNB1 (1:1000, Abclonal, A8610), β-ACTIN (1:1000, CST, 3700S), STAT3 (1:1000, Abclonal, A19566), P-STAT3 (1:1000, Abclonal, AP0705), VEGF (1:1000, Proteintech, 66828–1-Ig), P-VEGFR2 (1:1000, Cell Signaling Technology, #3770), GAPDH (1:1000, Abclonal, AC001), Laminb1 (1:1000, Cell Signaling Technology, #13435), ANGPT2 (1:1000, Abclonal, A11306), Flag (1:1000, Sigma, F2555), JAK1 (1:1000, Abclonal, A18323), JAK2 (1:1000, Abclonal, A7694), SRC (1:1000, Abclonal, A0324), TYK2 (1:1000, Abclonal, A2128), P-JAK1 (1:1000, Abclonal), GPX1 (1:1000, Abclonal, A11166), and MOV10 (1:1000, Abclonal, A7227).
Relative quantitative analysis of protein expression levels was performed using Image Lab software (Bio-Rad).
Tissue/cells on coverslips were fixed with 4% paraformaldehyde and deparaffinized. Antigen retrieval was carried out by heating. Then, the tissue sections/cells were incubated with donkey serum to block background signal, followed by the detection of specific antigens with primary and fluorescent secondary antibodies. The nuclei were stained with DAPI. Then the tissue sections/cells were observed and photographed under a fluorescence microscope (Olympus). Antibodies against the following were used: CD31 (1:200, Abcam, ab76533), KPNA2 (1:200, Abclonal, A5012), and P-STAT3 (1:200, Abclonal, AP0705). The fluorescent secondary antibodies used are as follows: Alexa Fluor488 donkey anti-rabbit lgG (H + L) (1:400, Life Technologies, A21206), Alexa Fluor555 donkey anti-rabbit lgG (H + L) (1:400, Life Technologies, A31572).
Recombinant adenovirus production
Recombinant adenovirus was constructed using Gateway cloning according to the manufacturer’s protocol. The entry vector for overexpression is the pENTY-ccDB-T2-pcdh vector with the CMV promoter, and the entry vector for knockdown is the PEnty-kd-ccDb2 vector with the U6 promoter. The Entry clone with the gene of interest flanked by attL sequences was produced using the restriction enzyme and ligase cloning method. Expression clones were generated with Gateway LR Clonas II enzyme mix (Thermo) catalysed in vitro by recombination of the entry vector (containing the gene of interest flanked by the attL sequences) with the destination vector (containing the attR sequences) pDEST (Thermo Scientific). Recombinant adenoviral plasmids were linearized with PacI (Thermo Scientific) and transfected into HEK293A cells for adenoviral packaging and amplification. Then, the adenovirus was purified by CsCl gradient centrifugation.
Tubule formation assay
Tubule formation assays were performed as described previously . A 96-well plate was coated with 50 µl of Matrigel per well, and then HUVEC were seeded in the Matrigel-coated 96-well plate at a density of 1.5 × 104 cells per well. After 6 h, pictures were taken with a light microscope (Olympus, Tokyo, Japan), and the Angiogenesis Analyser plugin of ImageJ was used to count the total length.
Migration assays were performed with a Transwell cell culture insert (3 µm, Corning, NJ). HUVEC (3 × 104 cells) were placed on the upper layer of a Transwell cell culture insert, and ECM was placed below the cell-permeable membrane. Following an incubation period (6 h), the cells that had migrated through the membrane were stained with 0.1% crystal violet for 20 min and counted .
5-Ethynyl-2′-deoxyuridine (EdU) staining
EdU experiments were performed with a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime Biotechnology, C0075S). Briefly, HUVEC were seeded in a 96-well plate at a density of 5000 cells per well. The cultured cells were labelled with EdU, fixed, washed, and permeabilized. Click reaction solution (Click Reaction Buffer, CuSO4, Azide 555, Click Additive Solution) was used for the EdU reaction and detection. DAPI was used to stain the nuclei. Fluorescence was observed under a fluorescence microscope (Olympus).
Extraction of cytoplasmic and nuclear proteins
HUVEC were prepared in 10 cm dishes for extraction of the cytoplasmic and nuclear proteins. Nuclear and cytoplasmic proteins were isolated using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents according to the manufacturer’s protocol (Thermo Scientific).
RNA extraction and quantitative RT‒PCR
RNA was extracted from tissues and cells using TRIzol (Takara, Japan). RNA (1000 ng) was reverse transcribed using a PrimeScript™ RT Reagent Kit (Takara, Japan). Quantitative RT‒PCR was performed with SYBR Green Master Mix (Vazyme, Nanjing, China). 18S rRNA was used as an endogenous control. Relative gene expression levels were calculated with the 2^(− ΔΔCT) method. The primers used were as follows:
KPNA2, forward 5′-CTGCCCGTCTTCACAGATTCA-3′, reverse 5′-GCGGAGAAGTAGCATCATCAGG-3′, ANGPT2, forward 5′-AACTTTCGGAAGAGCATGGAC-3′, reverse 5′-CGAGTCATCGTATTCGAGCGG-3′, VEGFA forward 5′-AGGGCAGAATCATCACGAAGT-3′, reverse 5′-AGGGTCTCGATTGGATGGCA-3′, 18S, forward 5′-TTGACGGAAGGGCACCACCAG-3′, reverse 5′-GCACCACCACCCACGGAATCG-3′.
Coimmunoprecipitation (Co-IP) assays
Co-IP assays were performed as described previously. The cells were lysed with RIPA lysis buffer (Beyotime Biotechnology, P0013C) supplemented with PMSF (Thermo Scientific) and protease inhibitors (Roche, 04693132001). The lysate was sonicated for 30 s and kept on ice for 1 h, and the supernatant was collected. Mixed 50 µl protein A/G magnetic beads (MedChemExpress, cat# HY-K0202) into the protein-antibody solution and incubated on a shaker at 4 °C for 3 h. The magnetic beads were precipitated with a magnetic stand. The magnetic beads were boiled with SDS loading buffer and subjected to immunoblotting analysis. The magnetic beads were also used for immunoprecipitation-mass spectrometry (IP-MS).
After sample processing, mass spectrometry data were collected using a Q Exactive Plus mass spectrometer (Thermo Scientific) in series with an EASY-nLC 1200 liquid phase LC/MS system (Thermo Scientific). The mass spectral data were searched with MaxQuant (V1.6.6) software, and the database search algorithm Andromeda was used to search the Human's Proteome Reference Database in UniProt (2020-05-10, containing 75074 protein sequences).
All data are presented as the mean ± SD. Statistical analysis between two groups was carried out by the two-tailed unpaired Student’s t test. All experiments were performed as multiple biological replicates (n = 3–6). Differences for which P < 0.05 were considered to be statistically significant. Statistical analysis was performed using GraphPad Prism 8.0.