HUVEC were obtained from ATCC (Manassas, USA) and cultured in Medium 199 (Invitrogen, Carlsbad, CA, USA) containing 10% (v/v) fetal bovine serum (FBS, HyClone, UT, USA) at 37 °C. For hypoxia experiments, cells were subjected to Earle’s solution (NaCl 116.4 mM, KCl 5.4 mM, CaCl2 1.8 mM, MgSO4 0.8 mM, NaH2PO4 2.6 mM, NaHCO3 26.2 mM, Hepes 20.1 mM, pH7.4)  saturated by 95% N2 plus 5% CO2 for 6 h in a Tri-gas hypoxia incubator (Forma Scientific, Marietta, OH, USA). ROS inhibitor, N-acetyl cysteine (NAC, #A0737), hydrogen peroxide solution (#323381) and cycloheximide (#5087390001) was purchased from Sigma-Aldrich (Steinheim, Germany). Paclitaxel (#S1150), colchicine (#S2284) and wortmannin (#S2758) were obtained from Selleck (Shanghai, China), Actinomycin D (#A4448) was purchased from Apexbio (Shanghai, China). PIK3CA-WT, PIK3CA-H1047R and E545K mutation were gifts from Joan Brugge (Addgene plasmid # 14572) . PI3K siRNA, stathmin1 siRNA and PIK3CA siRNA were designed and synthesized from GenePharm Co, Ltd. (Shanghai, China).
HUVEC migration was evaluated with transwell system (Corning Costar, MA, USA). Briefly, 1 × 105 HUVECs were seeded in the upper chambers for 12 h attachment. Then cells were subjected to hypoxic condition and chemical treatment for 6 h. The medium in upper chambers was switch to M199 with 0.5% FBS, while, that in the lower chamber were switch to M199 with 1% FBS. After 12 h incubation, the cells on the bottom were fixed with 4% paraformaldehyde and stained with 1% crystal violet, the non-migrating cells in the upper chamber were removed. Finally, the crystal violet was dissolved in 33% acetic acid, and the absorbance was measured at 600 nm. The amount of cell migration was determined as the ratio of the OD values of the treatment relative to the control. Each treatment was repeated in three independent chambers.
Trans-endothelial permeability assay
Cells were grown on 0.2 mm pore-size collagen IV (1 mg/mL)-coated tissue culture inserts (Nunc, Fisher Scientific, Pittsburgh, PA) until confluent. Monolayers were then serum-starved for 1 h and either left untreated or exposed to hypoxic condition in triplicate with desired agents. Following treatment, fluorescein isothiocyanate (FITC) dextran (10 kDa) dissolved in the medium was placed in the upper chamber at a concentration of 0.4 mg/mL and allowed to equilibrate for 2 h. Samples were then taken from the lower chamber for fluorescence measurements. Fluorescence was measured by excitation at 492 nm and the emission collected at 520 nm.
Transmission electron microscopy (TEM)
After treated, HUVECs were fixed for 1 h with cold 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) and washes with 0.2 M cacodylate, and then cells were pelleted and then postfixed with 2% osmium tetroxide in 0.1 M cacodylate for 1 h at 4 °C, stained en bloc with 2% uranyl acetate for an additional 1 h. After three more washes in double-distilled water, the samples were dehydrated in a series of acetone solutions and embedded in Epon 812 according to the standard procedure. Ultrathin sections (70 nm) were prepared, stained with both uranyl acetate and lead citrate, and assessed using a Hitachi 7400 electron microscope (Hitachi, Tokyo, Japan). Random fields taken from individual samples were photographed at 30,000×.
After hypoxia treatment, 5 μM dichlorofluorescein diacetate (DCF) was added into HUVEC cells culture medium and incubate for 1 h at 37 °C. Then cells were washed cells twice with PBS and scrape cells into RIPA buffer and incubate for 10 min on ice. After spin at 13,000 rpm for 10 min, 100 μl supernatant was transferred into 96-well plates for fluorescence assay (485 nm excitation and 527 nm emission). Protein concentration was tested for the normalization of cell number.
5-Bromo-2′-deoxy-uridine (BrdU) incorporation assays
HUVEC cells were subjected to hypoxic condition and chemical treatment for 6 h and then incubated with 0.1 mg/ml BrdU for 3 h before fixed by 4% paraformaldehyde (PFA). Cells were incubated in 0.5% Triton X-100, 1.5 N HCl, and washed by PBS between incubation, then add 0.25% trypsin EDTA, and incubate at 37 °C for 5 min. Cells were washed by PBS and blocked in blocking buffer for 2 h. BrdU antibody (1:100, ab6326, Abcam) was used and then incubated overnight at 4 °C in a hydration chamber. After wash with PBST, secondary antibody containing 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) were added and incubated at RT for another 3 h, then observed with Olympus IX81.
HUVEC cells were plated on fibronectin (10 µg/mL)-coated glass coverslips treated with indicated condition. Attached cells were fixed with 4%PFA, followed by permeabilization with 0.2% Triton X-100 at room temperature for 10 min, cells were stained with 100 nM Alexa Fluor™ 488-conjugated phalloidin (A12379, Thermo Fisher) in dark for 30 min and then incubated with DAPI at room temperature for 10 min. After wash with PBS, stained cells were examined by confocal microscope.
MTT assay for cell proliferation
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the effect of treatment on cell viability. HUVEC were inoculated into 96-well plates at 5 × 104 cells/ml, after treated with hypoxia and chemicals, 5 mg/ml MTT (Beyotime Biotech, Jiangsu, China) was added into the cell culture medium. After further 4 h incubation, the supernatant fraction was removed and 150 µl of dimethyl sulphoxide (DMSO) was added. The optical density (OD) at 490 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA).
Western blot analysis
Total lysates from HUVEC cells were obtained by lysing in RIPA buffer with protease inhibitors cocktail (#HY-K0010, MedChem Express, Shanghai, China). Protein concentration was measured by the BCA assay (Bio-Rad, Hercules, CA, USA). Proteins were extracted and separated in 10% Tris glycine/SDS–polyacrylamide gels and electro-transferred to ECL nitrocellulose membranes (#IPFL00010, Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk and incubated with specific antibodies overnight at 4 °C. β-Actin or P85 was used as the endogenous control. Primary antibodies were used at the dilution of 1:1000. Anti-Phospho-Akt (Ser473) (#4060), Akt (#4685), PI3K (#4249), stathmin1 (#13655) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin (ab8266), anti-P85 (ab191606), anti-α Tubulin (acetyl K40) (ab24610) and horseradish peroxidase-conjugated anti-mouse or rabbit IgG were purchased from Abcam (Cambridge, MA, USA).
Total RNA was extracted from cells using RNAiso Plus (Takara Bio Inc., Dalian, China) and was reverse-transcribed using M-MMLV Reverse Transcriptase (#A3500, Promega Biotech Co., Ltd, Beijing, China) according to the manufacturer’s protocols. RT-qPCR was performed using SYBR (#4385612, Thermo Scientific Inc.). Primer sequences used in the experiments were as follows: Stathmin 1 (homo) forward 5′-TCAGCCCTCGGTCAAAAGAAT-3′, reverse 5′-TTCTCGTGCTCTCGTTTCTCA-3′; PPIA (homo) forward 5′-CCCACCGTGTTCTTCGACATT-3′, reverse 5′-GGACCCGTATGCTTTAGGATGA-3′. The expression levels of mRNA were normalized to PPIA mRNA.
The data are expressed as mean ± SEM. All experiments were performed in triplicate. All statistical analyses were performed with the SPSS 19.0 using non-parametric tests. The Kruskall Wallis test followed by the Mann–Whitney test was used to detect differences between groups. P < 0.05 was considered statistically significant.