Male Fisher 344 rats were used for rat hepatocyte isolation. Male wild-type C57 black, PXR-null, and CAR-null mice were used for in vivo studies. All animals received humane care and were treated according to protocols approved by the University of Pittsburgh’s institutional animal care and use committee (IACUC).
Isolation and Culture of Hepatocytes
Rat hepatocytes were isolated by two-step collagenase perfusion, as previously described23
and then seeded at 3 × 106
cells per 100 mm of a collagen-coated dish in minimal essential media containing insulin. After 1 hour, the medium was changed to serum-free hepatocyte growth medium containing ITS and dexamethasone as described elsewhere.24
After 18–20 hours, cells were treated with PB (2 mmol/L unless otherwise indicated) and harvested at various times thereafter. When indicated, okadaic acid (10 nmol/L), KN62 (5 μmol/L), and RP isomer of 8-bromo-cyclic adenosine monophosphate (RP-8Br-cAMP; 10 μmol/L) were added 30 minutes prior to administration of PB.
RNA Isolation and Real-Time PCR Analysis
Total RNA was isolated using RNA-B solution (Tel-Test, Friendswood, TX), then treated with Turbo DNA-Free (Ambion, Austin, TX) according to the manufacturer’s instructions. Two micrograms of total RNA was reverse-transcribed with SuperScript II (Invitrogen, Carlsbad, CA). Forty nanograms of the resulting cDNA was used for Sybr Green real-time polymerase chain reaction (PCR) analysis (Applied Biosystems, Foster City, CA), along with mouse HNF-4α (SuperArray Biosciences, Frederick, MD) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers.25
Real-time PCR analysis was performed using an ABI Prism 7000 sequence detection system. Results were compiled from two independent experiments with assays performed three times with each sample in triplicate. Data presented are based on the comparative CT
method and reflect relative quantitation to GAPDH as the internal control. Statistical significance was determined using the Student t
Hepatocytes grown on collagen-coated coverslips were washed with (PBS), fixed with cold 2% paraformaldehyde in PBS for 15 minutes, permeabilized with 0.1% TritonX-100 in PBS for 15 minutes, washed five times in 0.5% BSA solution (PBS containing 20 mmol/L glycine and 0.5% BSA), then blocked for 1 hour with 2% BSA solution. Following five washes with 0.5% BSA solution, coverslips were incubated overnight with goat anti-HNF-4α, 1:500 (Santa Cruz Biotechnology, Santa Cruz CA), in 0.5% BSA solution. Cells were washed five times with 0.5% BSA solution, then incubated for 1 hour with donkey antigoat CY3 in 0.5% BSA solution. Following five washes in 0.5% BSA solution and then PBS, coverslips were incubated with Hoescht stain (30–45 s), washed with PBS, and mounted on slides. Fluorescence was visualized using an Olympus Provis fluorescence microscope at 60× magnification.
Cells from one 100-mm plate or 100 mg of liver tissue was lysed by sonication in RIPA buffer containing protease and phosphatase inhibitor cocktails (Sigma, St. Louis, MO), then incubated on ice for 30 minutes and centrifuged at 14,000g for 30 minutes. Supernatants were saved as total cell lysate.
For nuclear protein isolation, cells from 3–5 plates were washed and harvested in 40 mmol/L Tris (pH 7.6), 14 mmol/L NaCl, and 1 mmol/L EDTA, then pooled and centrifuged (5 min, 100g). Cell pellets were suspended in 2 mL of hypotonic buffer [10 mmol/L Hepes (pH 7.9), 10 mmol/L NaH2PO4, 1.5 mmol/L MgCl2, 1 mmol/L DTT, 0.5 mmol/L spermidine, and 1 mol/L NaF with protease and phosphatase inhibitor cocktails (Sigma, St. Louis, MO)]. Following 10 minutes of incubation on ice, samples were homogenized in a Dounce homogenizer and then centrifuged (5 min, 800g). Cell lysis was monitored with trypan blue stain. Supernatants were saved as cytoplasmic extracts. The nuclei pellets were washed two additional times in the same buffer. Mouse liver tissue (0.2–0.3 g) was directly homogenized in 5 mL of hypotonic buffer and then centrifuged (5 min, 100g). The pellets were resuspended in 2 mL of hypotonic buffer and processed as above.
Nuclear proteins were extracted in 50–100 μL of hypertonic buffer [30 mmol/L Hepes, (pH 7.9), 25% glycerol, 450 mmol/L NaCl, 12 mmol/L MgCl2, 1 mmol/L DTT, and 0.1 mmol/L EDTA with protease and phosphatase inhibitor cocktails (Sigma, St. Louis, MO)] for 45 minutes at 4°C with continuous agitation. Extracts were centrifuged at 30,000g, and the supernatants were collected and dialyzed for 2 hours against the same solution but containing 150 mmol/L NaCl. Protein concentration was determined by the Bicinchoninic Acid assay (Sigma, St. Louis, MO).
Western Blot Analysis
Nuclear proteins (20 μg), cytoplasmic extract (50 μg), or total cell lysate (100 μg) were separated by SDS-PAGE in 4%–12% NuPage Bis-Tris gels with 1× MOPS Buffer (Invitrogen, Carlsbad, CA), then transferred to Immobilon-P membranes (Millipore, Bedford, MA) in NuPAGE transfer buffer containing 0.005% SDS and 10% methanol. Membranes were stained with Ponceau S to verify loading and transfer efficiency. Membranes were probed with antibodies in TBST containing 5% nonfat milk, then processed with SuperSignal West Pico chemiluminescence substrate (Pierce, Rockford, IL) and exposed to blue X-ray film (Lab Product Sales, Rochester, NY). The primary antibodies used were: rabbit anti-HNF-4α N1.14 (F. Sladek, UC Riverside), 1:5,000; goat anti-HNF-4α C-19, rabbit anti-CAR H-150, and goat anti-PXR R14 (Santa Cruz Biotechnology, Santa Cruz CA), 1:500; and mouse anti-actin MAB150 (Chemicon, Temecula, CA) 1:2000. HRP-conjugated secondary antibodies were used at a 1:80,000 ratio (Chemicon, Temecula, CA). Protein expression was normalized to β-actin for total cell lysate or total protein per lane from Ponceau S stain of membrane for nuclear extracts.