Mesoglycan was kindly provided from Chodang Pham. CO.. Dulbecco’s modified eagle medium (DMEM) and fetal bovin serum (FBS) were purchased from Thermo Scientific (Logan, UT, U.S.A.). Pro-prep protein extract buffer was purchased from Intron Biotechnology (Sungnam, Korea). Antibodies against LKB1, phospho-LKB1 (Ser428), AMPK, phospho-AMPK (Thr172), phospho-ACC (Ser79), mTOR, phospho-mTOR (Ser2448), Bcl-2, Bax, phospho-p70S6K (Thr389) and phospho-4EBP1 (Ser65) were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). A monoclonal antibody against β-actin was purchased from Sigma-Aldrich (St. Louis, MO). Compound C, AMPK inhibitor, was provided by Calbiochem (La Jolla, CA, U.S.A.). Human Platelet-Derived Growth Factor BB was purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). AMPK siRNA, Raptor siRNA, Control siRNA, p53, p27, p21, PCNA and Cytochrome C were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.).
Sprague–Dawley rats (SD rats) were anesthetized with pentobarbital (50 mg/kg). Vascular smooth muscle cells (VSMCs) were isolated from thoracic aorta and the connective tissue was removed. Aortic VSMCs were grown in DMEM with 10 % FBS and 1 % antibiotic (penicillin 10000 U/ml). It was processed using a 1 mm chop setting in a 10 cm culture dish, and cultured with 50 % FBS-DMEM with 1 % antibiotics and incubated in a CO2 incubator (95 % CO2 air, 37 °C). We used VSMCs from 6 to 8 passages at 70–90 % confluence in 10 cm dishes, and cell growth was arrested by incubation of the cells in serum-free DMEM for 24 h prior to use.
Western blot analysis
Whole cell extracts were prepared by lysing the cells in pro-prep protein extract buffer. The protein concentration was quantified with protein assay reagent from Bio-Rad (Hercules, CA, U.S.A.). Equal amounts of protein were mixed with Laemmli Sample Buffer (Bio-Rad) and heated for 5 min at 100 °C before loading. Total protein samples (30 μg) were subjected to 10 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for 1 h 30 min at 100–120 V. The separated proteins were electrophoretically transferred onto a PVDF membrane for 1 h 20 min at 100 V using SD Semi-dry Transfer Cell. The membranes were blocked with 5 % non-fat milk in PBS containing 0.05 % Tween 20 (PBS-T) for 1 h at room temperature. The membranes were then incubated with the primary antibodies at a dilution of 1:1000 by overnight at 4 °C in PBST. The membranes were then washed with four changes of wash buffer (0.05 % Tween 20 in PBS) and incubated for 1 h at room temperature in PBS containing anti-rabbit(Stress-gen, Ann Arbor, MI, USA) and anti-mouse IgG (Sigma, St. Louis, MO, USA) antibodies. Finally, after three more rinses with wash buffer, the membranes were exposed to ECL and ECL Plus western blot analysis detection reagents.
Transfection of siRNA
Transfection of VSMCs with siRNA was performed using lipofectamine-2000 reagent, according to the manufacturer’s instructions. Aliquots of 1 × 104 cells were plated on 6 well on the day before the transfection and grown to about 70 % confluence. The cells were then transfected with 10 μM AMPK siRNA (Santa Cruz Biotechnology Inc., Santa Cruz, CA) + 100 pmol of Lipofectamine for 6 h in Opti-MEM®I reduced serum medium (Invitrogen, Carlsbad, CA, USA). Following an incubation period of 48 h, the AMPK protein level was measured using western blot analysis, and cell proliferation was analyzed using the MTT assay.
Adenoviruses expressing the control gene GFP, the dominant-negative isoform of the α1 and α2 subunits of AMPK (AMPK DNα1 and DNα2) were amplified in AD293 cells using standard methodologies. The transductions were carried out in VSMC in sereum-fress DMEM for 6 h.
Flow cytometric analysis for apoptosis and cell cycle
Apoptosis was examined by Annexin V-fluorescein isothiocyanate (FITC) staining (BD Biosciences, San Jose, CA, U.S.A.) according to the manufacturer’s instructions. Cells were seeded on 6-well plates and incubated for 2 days. Cells were treated with mesoglycan (0.1 μg/ml) for 24 h. The FITC fluorescence intensity of 10,000 cells was measured using a Becton-Dickinson FACS Caliber flow cytometer (BD Biosciences). Cell cycle profiles were analyzed by propidium iodide (PI) staining. A minimum of 10,000 cells in each sample was detected according to intracellular PI fluorescence intensity by flow cytometry, and cell cycle was analyzed by Cell Quest software (BD Biosciences).
Fluorescence intensity measurements
Cells were washed and then maintained in complete medium. After detachment from dishes with 50 mM EDTA, the cells were centrifuged at 3000 rpm for 10 min and resuspended in PBS containing 2 % bovine serum albumin. After labeling, cells were washed once in PBS, fixed in 4 % paraformaldehyde, and analyzed on a flow cytometer. For each sample, 1000 cells were analyzed, and the results were expressed as geometric mean fluorescence.
VSMCs were seeded on coverslips in 35 mm glass bottom dishes, fixed in 4 % formaldehyde, and permeabilized with 0.2 % Triton X-100. The p-mTOR primary antibody was used at 1:100 (Santa Cruz Biotechnology) and incubated with cells overnight at 4 °C. Rabbit FITC secondary antibody (Invitrogen) was used at 1:100 and incubated with cells for 1 h at room temperature. Fixed and immunofluorescently stained cells were imaged using a Leica confocal microscope (Bannockburn, IL, USA).
Cell proliferation assay
VSMCs were seeded on 24-well plates at 1 × 104 cells per well in DMEM supplemented with 10 % FBS. After different treatments, 50 μl of 1 mg/ml MTT solution was added to each well (0.1 mg/well) and incubated for 4 h. The supernatants were aspirated, and the formazan crystals in each well were solubilized with 200 μl dimethyl sulfoxide (DMSO). An aliquot of this solution (100 μl) was placed in 96-well plates. Cell proliferation was assessed by measuring the absorbance at 570 nm using a microplate reader.
Results are expressed as mean ± SEM from at least three independent experiments. Differences between data sets were assessed by one-way analysis of variance (ANOVA) or Bonferroni's test.