Reagents and cell lines.
Human 143B osteosarcoma cells were obtained from the American Type Culture Collection and cultured according to the vendor's recommendations. Murine embryonic fibroblasts (MEFs) from mice genetically deficient for LKB1 (LKB1−/−
) were obtained as a gift from Ronald DePinho (1
) and cultured in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Adenovirus expressing the calcium-sensitive fluorescence resonance energy transfer (FRET) probe YC2.3 (13
) was obtained as a gift from Michael Roe. An antibody for β-actin was obtained from Abcam, and antibodies for CaMKKβ were obtained from Santa Cruz Biotechnology and Sigma. All other antibodies were from Cell Signaling Technology. Short interfering RNAs (siRNAs) were obtained from Ambion. Other chemicals and reagents were obtained from Sigma.
Cells grown to 70 to 80% confluence on 25-mm glass coverslips were placed into a flowthrough chamber consisting of two coverslips separated by a stainless steel spacer ring. In the chamber, cells were superperfused with balanced salt solution (BSS) bubbled with O2-CO2-N2 gas mixtures at 37°C in a water-jacketed column. Cells were equilibrated at baseline with a BSS containing NaCl (117 mM), KCl (4 mM), NaHCO3 (18 mM), MgSO4 (0.76 mM), NaH2PO4 (1 mM), CaCl2 (1.21 mM), and glucose (5.6 mM) bubbled with 5% CO2, 21% O2, and the balance N2. For hypoxia, BSS was bubbled with 5% CO2, 1.5% O2, and the balance N2. Trace calcium BSS contained CaCl2 at 0.1 mM, while calcium-free BSS was made without CaCl2 and included EGTA (2.5 mM) with the final pH adjusted to 7.4.
Western blot analysis.
At the end of treatment with hypoxia, the tubing at each end of the flowthrough chamber was clamped and the sealed chamber was submerged in an ice-water slurry to chill cells quickly. The chamber was then opened, and the cells were dissociated with a lysis buffer consisting of Tris HCl pH 7.4 (50 mM), NaCl (150 mM), Triton X-100 (1%), EDTA (2 mM), β-glycerophosphate (40 mM), phenylmethylsulfonyl fluoride (PMSF) (1 mM), NaF (10 mM), sodium orthovanadate (250 μM), and a protease inhibitor cocktail (Roche). Cells in lysis buffer were scraped from the coverslips, flushed several times through a tuberculin syringe, and incubated on ice for 15 min. Whole-cell lysates were centrifuged at 16,000 × g for 10 min and supernatants collected. Lysates were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes that were blotted with primary antibodies. Blots were further incubated with secondary horseradish peroxidase (HRP)-conjugated antibodies and stained with ECL reagent (Amersham). Chemiluminescence was detected on film and quantified using Image J.
Assessment of oxidant signaling using roGFP or DCFH and of calcium using the YC2.3 sensor.
A recombinant adenovirus expressing the redox-sensitive green fluorescent protein (roGFP) redox sensor (9
) was utilized to achieve high transduction efficiency in cultured cells. Other cells were transduced with recombinant adenovirus expressing YC2.3, a ratiometric calcium indicator (13
). Infected cells were returned to the incubator for 24 to 36 h to allow for protein expression. For imaging experiments, cells were superperfused with BSS under controlled O2
conditions in a flowthrough system as described above. Images were collected using a 16-bit cool charge-coupled device (CCD) detector, using excitation wavelengths of 400 and 485 nm (roGFP) or 430 nm (YC2.3). Fluorescence was detected at 535 nm (roGFP) and at 470 and 535 nm (YC2.3). Ratios for roGFP (485/400) or YC2.3 (535/470) were assessed using Metafluor software (Universal Imaging).
Oxidant signaling was also assessed using 2,7-dichlorofluorescin diacetate (DCFH-DA). Cells were superfused with BSS containing DCFH-DA (5 μM), and fluorescence images were collected every minute at 535 nm using excitation at 484 nm. Cellular fluorescence data were collected from individual cell regions of interest and averaged to provide an overall assessment for each coverslip. Values obtained from replicate coverslips were then averaged.
Knockdown of gene expression with siRNA.
Cells grown on coverslips were transfected with siRNA (100 nM) using Lipofectamine RNAmax reagent (2.5 μl) in a final volume of 0.7 ml Optimem (Invitrogen). After incubation (5 h) with occasional rocking, 1.5 ml of complete medium was added. The medium was changed after an overnight incubation, and cells were analyzed at 48 h posttransfection. Control cells were transfected with a negative-control siRNA. The following siRNA oligonucleotides from Ambion were used in this study: negative-control 2 siRNA, STIM1 siRNA s13561, CaMKKβ siRNAs s101668 and s20926, and LKB1 siRNA s74499. Knockdown of gene expression was quantified by Western blotting.
Nifedipine was diluted in dimethyl sulfoxide (DMSO) and used at a final concentration of 2 μM. Cells were superperfused with BSS containing nifedipine or DMSO under controlled O2/CO2 conditions in a flowthrough system as described above. Lysates were collected and analyzed by Western blotting.
Measurement of adenine nucleotide concentrations.
ATP, ADP, and AMP concentrations were determined by high-performance liquid chromatography (HPLC) (27
). After incubation inside a glove box (Coy Laboratories) equilibrated to 1.5% O2
, 5% CO2
, and the balance N2
for 2 h, 3 × 107
cells were immediately harvested in a minimal volume of ice-cold 66.7 mM perchloric acid and spun down in a refrigerated centrifuge for 10 min at maximum speed. After centrifugation, 10 μl of 2 M Tris (pH 8.0) was added to 300 μl of supernatant, and the extracts were neutralized with 10 μl of 1N KOH. Samples were analyzed by HPLC as previously described (3
143B human osteosarcoma cells on coverslips were treated with a single bolus of tert-butyl hydroperoxide (tBH) (100 μM) or thapsigargin (TG) (2 μM) or with brief hypoxia (1.5% O2) and fixed with 3% formaldehyde in phosphate-buffered saline (PBS). Cells were permeabilized with ice-cold methanol and blocked using 1% normal goat serum (NGS) in PBS. Cells were hybridized with a primary antibody specific for STIM1 diluted in 1% NGS in PBS at 1:100 followed by an Alexa anti-rabbit secondary antibody diluted 1:1,000. Cells were imaged on a Zeiss LSM 510 META laser confocal system.
Fura2-AM hypoxia studies.
143B human osteosarcoma cells on coverslips were incubated with fura-2-acetoxymethyl ester (Fura2-AM) (5 μM) for 1 h under normal culture conditions and then incubated for 15 min longer in BSS without dye. For imaging experiments, cells were superperfused with BSS under controlled O2/CO2 conditions in a flowthrough system as described above. Images were collected using a 16-bit cool CCD detector, using excitation wavelengths of 340 and 380 nm. Fluorescence was detected at 535 nm, and ratios (340/380) were assessed using Metafluor software (Universal Imaging).
Formalin-fixed, paraffin-embedded 4-μm sections of mouse lungs were placed on charged slides. Lung sections were deparaffinized, and target retrieval was carried out using a digital pressure cooker (Biocare Medical) heated to 125°C for 30 s and then cooled to 90°C before venting. Hydrogen peroxide (3%) was applied to the sections for 10 min to quench endogenous tissue peroxidase. The lung sections were incubated with primary antibodies specific for ACC or for ACC phosphorylated at Ser-79 (p-ACC) (Cell Signaling Technology), according to the manufacturer's recommendations, and with Dako anti-rabbit secondary antibodies. Immunohistochemical reactions were developed with diaminobenzidine and counterstained in Mayer's hematoxylin (Sigma). Stained lung sections were imaged on a Zeiss Axioskop system equipped with a CRi Nuance spectral camera. Images were color separated using Nuance software. Blue and brown layers were analyzed with Metamorph software to calculate the integrated pixel intensity for the stained areas in each field. The total integrated intensity for p-ACC or ACC staining in each image was normalized to hematoxylin stain.
Mouse hypoxia experiments.
Mice were housed in room air or 10% O2
in an environmentally controlled glove box for 28 days. Some mice housed under hypoxia were treated with the chemical antioxidant N
-cysteine (NAC) administered in their drinking water at a final concentration of 40 mM (12
). NAC-supplemented drinking water was prepared and changed daily. Animal studies were approved by the Northwestern University IACUC.
Pulmonary microvessel myocyte isolation.
Rat pulmonary arterial smooth muscle cells (PASMC) were isolated as described previously (50
) using a modification of the method of Marshall et al (35
). Cells isolated by this method were confirmed to be PASMC as previously described (50
Ca2+ imaging studies.
143B cells were grown on poly-d-lysine-coated glass-bottom dishes (MatTekcorp) and loaded with 2.5 μM Fura2-AM (Invitrogen) for 35 min at room temperature. Cells were treated with thapsigargin (1 μM; Sigma) in 0 mM Ca2+ Ringer solution (150 mM NaCl, 4.5 mM KCl, 10 mM d-glucose, 5 mM HEPES, 1 mM EGTA, and 3 mM MgCl2) to deplete endoplasmic reticulum (ER) Ca2+ stores, and 2 mM Ca2+ Ringer solution (150 mM NaCl, 4.5 mM KCl, 10 mM d-glucose, 5 mM HEPES, 1 mM EGTA, 1 mM MgCl2, and 2 mM CaCl2) was then added to isolate store-operated Ca2+ influx. The same protocol was used to study intracellular Ca2+ responses to tBH. Fura images were acquired by alternately exciting Fura2-loaded cells at 340 nm and 380 nm and collecting the emission at 510 nm. Image pairs were acquired every 6 s on an IX71inverted microscope (Olympus, Center Valley, PA) equipped with a 40× oil-immersion objective, a xenon arc lamp (Sutter, Novatao, CA), and excitation and emission filter wheels (Sutter). Images were captured on a cooled CCD camera (Hamamatsu). Image acquisition and analysis were performed with IPLab software (Scanalytics, Rockville, MD). Background-subtracted images were thresholded, and regions of interest were drawn around single cells. The 340/380 intensity ratio was plotted as a function of time.