3.1. Primary hepatocytes isolation and culture
Primary hepatocytes were harvested from six-week old male C57BL/6 mice (BioLASCO) with following procedure. Briefly, mice were anesthetized and their portal vein were first perfused with PBS followed by Liver Perfusion Medium (Life Technology) and Liver Digestion Medium (Life Technology). Perfused liver was minced, suspended with Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS, GeneDireX) and 1% penicillin/streptomycin/glutamine (PSG, Life Technologies) and passed through a 40-μm cell strainer (BD Falcon). Cells were washed several times with DMEM and re-suspended with primary cell culture medium [DMEM supplemented with 10% FBS, 1% PSG, 1% glutamine (Life Technologies), 1% ITS (Life Technologies), 1% NEAA (Life Technologies) and 0.1% dexamethasone (Life Technologies)]. Cells were then centrifuged through a discontinuous Percoll (GE Healthcare) gradient and cultured on 0.1% gelatin-coated dish at 37oC in a humidified incubator with 5% CO2. Hypoxic culture was conducted with same culture medium in the incubator with 5% CO2 and 1% O2.
3.2. Cellular RNA extraction
Cellular RNA was extracted with TriPure isolation reagent (Roche). 200 µL
chloroform was added into 1 mL cell extraction. The extraction was mixed by vortexing for 15 seconds and centrifuged at 12000 g at 4oC for 15 minutes. RNA was extracted and then was precipitated using 0.7x aqueous phase volume isopropanol, followed by centrifugation at 12000 g at 4oC for 10 minutes.
Precipitated RNA was washed twice with 70% ethanol and air-dried. After dissolving in 30 µL DEPC/ddH2O at 55oC for 15 minutes, the quality and concentration of RNA was determined by Nanodrop 2000 (Thermo).
3.3. Quantitative reverse transcription polymerase chain reaction (qRTPCR) 2 µg total RNA was reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystem) according to the protocol manual. For quantification, 1 ng cDNA was used for qPCR with 200 nM forward and reverse primers in total 10 µL reaction volume by SYBR Green system Rad). qPCR was performed using Bio-Rad CFX96 Real-Time PCR Machine (Bio-Rad) with following protocol: denaturation at 95oC for 3 minutes, 40 cycles of elongation at 65oC for 30 seconds and denaturation at 95oC for 10 seconds. The expression level of target genes was assayed in triplicate and normalized to internal control. The primer used for qPCR were listed in Table 1.
3.4. Cellular protein extraction
buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA) supplemented with 1X cOmplete Cock Tail Protease Inhibitor (Roche) on ice. The total cell lysates were centrifuged at 12000 g at 4oC for 10 minutes to remove the pellet. Protein concentration was quantified by Protein Assay Dye Reagent (Bio-Rad). Protein samples were denatured with 5x protein sample buffer (50% glycerol, 10% SDS, 0.25 M Tris-HCl, 0.05% Bromophenol blue, 5% β-mercaptoethanol) at 100oC for 10 minutes.
3.5. Western blotting
Protein samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE gel (10% separation gel and 4 % stacking gel) and TGS buffer system (50 mM Tris-HCl, pH 8.0, 380 mM Glycine, 0.1% SDS) were used to separate total protein. Electrophoresis was performed by following protocol: 100 V for 20 minutes and 150 V for 1.5 hour. Proteins were then transferred onto PVDF membrane (Roche) by Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad) at 1.5 mA/cm2 for 90 minutes. The membrane was washed with TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% Tween-20) and blocked with 4% skim milk in TBST for 1 hour at room temperature. After washed with TBST, primary antibody [diluted in gelatin-NET (0.25% gelatin, 0.15 M NaCl, 5 mM EDTA, 0.05% Tween-20, 50 mM Tris-HCl ,pH 8.0) or 4% skim milk] was added
and incubated in 4oC overnight. The membrane was washed thrice and second antibody (diluted in gelatin-NET) was added for 1 hour at room temperature. After washed with TBST twice and once in ddH2O, membrane was rinsed with LuminateTM Classico Western HRP Substrate (Millipore) or ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore) for 1 minute and analyzed by Biospectrum MultiSpectral Imaging System (UVP BioSpectrum 800). The antibodies and their dilution condition were listed in Table 2.
3.6. Cell immunofluorescence staining
Cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 10 minutes and then permeabilized with 0.5% Triton X-100 at room temperature for 10 minutes. After blocked with 3% FBS (diluted in PBS) at room temperature for 1 hour, cells were incubated with primary antibody at 4oC overnight. Second antibody was added and incubated for 1 hour at room temperature. At last, 5 µg/mL Hoechst 33342 was added for nucleus staining and incubated for 10 minutes. The cells were stored in PBS at 4oC before analyzed by IN Cell 2000 (GE Healthcare). The antibodies and their dilution condition were listed in Table 2.
3.7. Luciferase assay
Cells were transfected with pGL4.10[luc2] containing EpCAM promoter and
PBS and lysed in 200 µL lysis buffer (10% glycerol, 2 mM EDTA, 1% Triton X-100, 2 mM DTT, 25 mM Tris-HCl, pH 7.4) with vigorous scraping. For luminescence measurement, 20 µL of cell lysates were transferred to white 96-well microplate and then the measurement was performed using Orion L Microplate Luminometer (Berthold Detection Systems). Fluorescence intensity of mCherry was measured by FlexStation 3® microplate reader (Molecular Devices) for internal control.
3.8. Chromatin immunoprecipitation (ChIP)
Cells were fixed with 1% paraformaldehyde (PFA) at room temperature for 10 minutes and were neutralized by addition of 125 mM Glycine. Cells were rinsed with cold PBS and collected by scraping with 1 mL Farnham lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% NP-40). Cells lysates were centrifuged at 800 g at 4˚C for 5 minutes and the pellets were collected. After re-suspending the pellets with Farnham lysis buffer, they were broken by passing through 20 G needle for at least 20 times. Nuclei were collected by centrifugation at 800 g at 4˚C for 5 minutes and re-suspended in RIPA buffer (1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS).
Cells were then sonicated with Covaris S2 (Covaris) to break the DNA into 500 bp-fragments. For immunoprecipitation, chromatin was first precleared by Protein A/G PLUSAgarose (Santa Cruz) at 4˚C for 30 minutes. After centrifugation at 12000 g
at 4˚C for 5 minutes, agarose beads were removed. Primary antibody was then added into the sample and incubated at 4˚C overnight. Agarose beads were added into the sample and incubated at 4˚C for 2 hours and harvested by centrifugation at 12000 g at 4˚C for 20 seconds. After washed with RIPA buffer twice and wash buffer (100 mM Tris-HCl, pH 8.0, 500 mM LiCl, 1% NP-40, 1% Na deoxycholate) for four times, agarose beads were re-suspended with 200 μL elution buffer (1% SDS, 100 mM NaHCO3). Samples were vortexed and incubated at 67˚C for 2 hours with occasional inverting. Agarose beads were removed by centrifugation at 10000 g at 4˚C for 3 minutes and the residuals were incubated at 67˚C overnight to perform reverse cross-linking. At last, DNA fragments were extracted by QIAquick PCR purification kit (QIAGEN) and the DNA concentration was determinated by Qubit (Life Technology). Input samples were prepared by incubation at 67˚C overnight to perform reverse cross-linking. After incubated with proteinase K for 10 minutes at room temperature and addition of RNase A, DNA was harvested by QIAquick PCR purification kit.
3.9. Image processing
Immunofluorescence images were obtained by IN Cell 2000 (GE Healthcare) and the background were subtracted before merging channels. Fluorescence
3.10. Statistical analysis
qRTPCR data show mean ± SEM. Statistical significance was calculated using a two-tailed Student’s t-test. Luciferase assay data show mean ± SD. Statistical significance was calculated using two-way ANOVA followed by Tukey’s multiple comparisons test. P values less than 0.05 were considered statistically significant.