Reagents and Cell Culture
Antibodies against Akt and phospho-Akt Ser473 and Thr308 were obtained from Cell Signaling Technology, Inc. (Beverly, MA). Antibodies for collagen type I and type III were from Fitzgerald (Concord, MA). Antibodies for HIF-1α, β-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Human VEGF immunoassay kit, a neutralizing antibody against VEGF, and its nonspecific control antibody were from R&D Systems (Minneapolis, MN). LY-294002, wortmannin, PD98059, and Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) were obtained from Calbiochem (La Jolla, CA). Catalase was from Roche Molecular Biochemicals (Indianapolis, IN). Akt mutant plasmid (SRα-Akt T308A/S473A) and control plasmid were a gift from C. Huang (New York University, Tuxedo, NY). VEGF reporter (VEGF-luc) and control plasmid were obtained from American Type Culture Collection (ATCC, Manassas, VA). All other chemicals and reagents including bleomycin sulfate, dihydroethidine, and dichlorofluorescein diacetate were from Sigma Chemical, Inc. (St. Louis, MO).
The human lung fibroblast CRL-1490 cell line was obtained from ATCC. The cells were maintained in Eagle's Minimum Essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. They were cultured at 37°C in 5% CO2 incubator. The cells were passaged at preconfluent densities using a solution containing 0.05% trypsin and 0.5 mM EDTA.
Cell Proliferation and Collagen Assays
Cells were seeded in 12-well plates at a density of 2 × 105 cells/well in MEM supplemented with 10% FBS at 37°C in a 5% CO2 incubator. At various times after the treatment, cells were scraped and washed twice with phosphate-buffered saline (PBS) and centrifuged at 1,000 × g for 5 minutes. Cells were resuspended in 1 ml of Hanks' balanced salt solution and counted using a hemocytometer. A minimum of three separate experiments was performed for each assay. Collagen content was determined by Western blotting as described below and by Sircol assay (Biocolor Ltd, Belfast, UK), according to the manufacturer's protocol. Briefly, Sirius red reagent (50 μl) was added to cell culture supernatant (50 μl) and mixed for 30 minutes. The collagen–dye complex was precipitated by centrifugation at 16,000 × g for 5 minutes, washed with ethanol, and dissolved in 0.5 M NaOH. The samples were introduced into a microplate reader and read for absorbance at 540 nm.
Apoptosis was determined using an enzyme-linked immunosorbent assay (ELISA)-based DNA fragmentation assay kit (Roche Molecular Biochem., Indianapolis, IN), according to the manufacturer's instructions. Briefly, cells were lysed with 200 μl of lysis buffer at room temperature, and the cell lysate (20 μl) was mixed with an antibody solution (80 μl) at room temperature for 2 hours. The substrate was then added after the wells were washed three times with a washing buffer. After incubation for 15 minutes at 37°C, optical density was measured using a microplate reader at the wavelength of 405 nm.
Western Blot Analysis
After specific treatments, cells were harvested and lysed on ice for 30 minutes in lysis buffer containing 150 mM NaCl, 100 mM Tris (pH 8.0), 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 5 mM EDTA, 10 mM sodium formate, 1 mM sodium orthovanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 2 mM pepstatin A. After centrifugation at 14,000 × g for 15 minutes at 4°C, the supernatant was collected as the total cellular protein extract. The protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL). Equal amount of proteins per sample (20 μg) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked with T-PBS (0.3% Tween-20 in PBS) containing 3% dry milk and incubated with primary antibody overnight at 4°C. After three washes with T-PBS, the membrane was incubated with HRP-conjugated secondary antibody for 1 hour and then washed with 0.05% Tween-20 in PBS. Immunoreactive proteins were detected by chemiluminescence (Supersignal West Pico; Pierce, Rockford, IL) and quantified by imaging densitometry using UN-SCAN-IT digitizing software (Silk Scientific, Orem, UT). Mean densitometry data from independent experiments were normalized to results in cells from control experiments.
Cellular ROS production was determined fluorometrically using dihydroethidine (DHE) and dichlorofluorescein diacetate (DCF-DA) as fluorescent probes for superoxide and peroxide, respectively. After specific treatments, cells were incubated with the probes (10 μM) for 30 minutes at 37°C, after which they were washed, resuspended in PBS, and analyzed for fluorescence intensity using a multiwell plate reader (FLUOstar OPTIMA; BMG LABTECH Inc., Durham, NC) at the excitation/emission wavelengths of 485/535 nm and 485/610 nm for DHE and DCF fluorescence measurements, respectively.
Stable Transfection of Dominant-Negative Akt
CRL-1490 cells were cultured in a 6-well plate until they reached 70 to 80% confluence. The cells were transfected with 1 μg of CMV-neo vector and 15 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) along with 2 μg of mutated Akt (SRα-Akt T308A/S473A) or control plasmid in the absence of serum. After 5 hours, the medium was replaced with 5% FBS MEM, and 36 hours later they were trypsinized and plated onto 75-ml culture flasks. The cells were then cultured in G418 selection medium (400 μg/ml) for 28 days. The selected cells were grown in G418-free MEM for two passages before each experiment.
VEGF Protein and Reporter Gene Assays
For analysis of VEGF protein, cells were plated in a 6-well plate at a density of 2 × 105 cells/well in culture medium and incubated overnight before the cells were subjected to treatment. After the treatment, cell supernatants were collected and analyzed for VEGF protein levels using a Quantikine ELISA kit (R&D Systems). Cell samples or reference standards (200 μl) were added to the wells of a microplate that was pre-coated with a monoclonal antibody specific to VEGF and incubated for 2 hours at room temperature. After washing unbounded substances, an HRP-conjugated polyclonal antibody against VEGF was added to the wells and incubated for 2 hours at room temperature. After washing and addition of 200 μl of substrate solution, optical density was determined on a microplate reader at 450 nm.
For VEGF reporter gene assay, cells were seeded in 12-well plates and cultured to 50 to 60% confluence. Cells were transfected with 1 μg of either VEGF reporter (VEGF-luc) plasmid (ATCC) or control plasmid and 10 ng of pRL-tk normalizing luciferase plasmid (Promega, Madison, WI) using Lipofectamine 2,000 transfecting agent (Invitrogen), according to the manufacturer's protocol. After a 24-hour recovery period, transfected cells were treated with bleomycin for 12 hours and cell extracts were prepared and analyzed for luciferase activity using a Promega dual-luciferase assay kit (Promega). Both firefly and Renilla luciferase activities were measured using a multiwell plate luminometer (BMG LABTECH). Normalized reporter activity was expressed as the firefly luciferase value divided by the Renilla luciferase value. Relative fold activity was calculated as the normalized reporter activity of the treated sample over control.
Reverse Transcription PCR
Total RNA was extracted with TRIZOL (Invitrogen) and reverse transcription PCR was performed with Access RT-PCR System (Promega) according to the manufacturer's instructions. The thermal profiles consisted of 94°C for 3 minutes for initial denaturing followed by 30 cycles of 95°C for 1 minute, 64°C for 1 minute, and 72°C for 1 minute. Specific primers used for PCR are as follows: VEGF (forward, 5′-TCTTCAAGCCATCCTGTGTGC-3′; reverse, 5′-CACATTTGTTGTGCTGTAGGA AGC-3′), HIF-1α (forward, 5′-TGTAATGCTCCCCTCACCCAACGAA-3′; reverse, 5′-CAGGGC TTGCGGAACTGCTTTCTA A-3′), and glyceraldehyde-3-phosphate dehydrogenase (forward, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′; reverse, 5′-AGCCTTCTCCATGGTTGGTGAAGAC-3′). The PCR products were electrophoresed in a 1.5% agarose gel, stained with ethidium bromide, and photographed.
Inhibition of HIF-1α by RNA Interference
Lentiviral transduction particles carrying short hairpin RNA (shRNA sequence against human HIF-1α (5′-CCGGCGGCGAAGTAAAGAATCTGAACTCGAGTTCAGATTCTTTACTTCGCC GTTTTT-3′) and control non-target sequence (5′-CCGGCAACAAGATGAAGAGCACCAACTC GAGTTGGTGCTCTTCATCTTGTTGTTTTT-3′) were used to knockdown HIF-1α expression in CRL-1490 cells. The viral vectors were obtained commercially from Sigma Chemical, Inc. (Cat # SHVRS-NM_001530 and SHC002V, respectively) and were used according to the manufacturer's instructions. Briefly, cells were seeded in 6-well plates (5 × 105/well) and incubated with HIF-1α shRNA lentiviral particles or control particles at the multiplicity of infection (MOI) of 1.5 in the presence of hexadimethrine bromide (8 μg/ml) for 36 hours. Transfected cells were analyzed for HIF-1α by Western blotting before use.
The data represent mean ± SD from three or more independent experiments. Statistical analysis was performed by ANOVA and Duncan's comparison tests were used to evaluate the significance between measurements at a significance level of P < 0.05.