HuH-7 cells were obtained from RIKEN cell bank (Ibaraki, Japan). Cells were cultured in Dulbecco's Modified Eagles Medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillin (final concentration, 100 U/ml), and streptomycin (final concentration, 0.1 mg/ml), in a humidified atmosphere of 5% CO2 and 95% air at 37°C.
Coomassie Brilliant Blue staining
HuH-7 cells were lysed in a lysis buffer [50 mM Tris–HCl, 150 mM NaCl, 0.1% (w/v) sodium deoxycholate, 0.1% (v/v) Triton X-100, and protease inhibitor cocktail, pH 7.4] after 4-h treatment with and without adenosine (10 mM). Then, lysates were loaded on 10% (v/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and separated proteins were stained with Coomassie Brilliant Blue.
Plasmid construction and transfection
Nucleotide sequence coding for AMID was cloned into pEGFP-Cl vector (Clontech) at the XhoI and EcoRI site. The plasmid DNA for AMID was transfected into HuH-7 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) or an electroporation system (Optimizor500, BTX, San Diego, USA).
AMID siRNA transfection
The siRNA silencing the AMID-targeted gene, that was purchased from Cosmo Bio (Kyoto, Japan), was transfected using a Nucleofector II device (Amaxa Biosystems, Cologne, Germany).
Total cellular RNA from HuH-7 cells was prepared using a Sepasol-RNA I Super kit. The first-strand cDNAs were synthesized using a High-Capacity cDNA Archive Kit. Each cDNA (2 μl) was amplified in a SYBR Green Realtime PCR Master Mix (final volume, 20 μl) and loaded on the Applied Biosystems 7900 Real-time PCR Detection System (ABI, Foster City, USA). Thermal cycling conditions were as follows: the first one step, 94°C for 4 min and the ensuing 40 cycles, 94°C for 1 s, 65°C for 15 s, and 72°C for 30 s. A standard curve was made by amplifying 0.5, 1, 2, 4, and 8 μl of the GAPDH cDNA diluted at 1:250. mRNA quantity for each target gene was calculated from the standard curve using an SDS 2.1 Software (Applied Biosystems, Foster City, USA). The levels for the AMID and AIF mRNA were normalized by the GAPDH mRNA. The primers as shown in Table 1 were used for real-time RT-PCR.
HuH-7 cells expressing HA-AMID were treated with adenosine (10 mM) for 4 h. Subsequently, cells were fixed with methanol and acetone at −20°C, each for 10 min, blocked with 10% (v/v) goat serum in phosphate buffered saline (PBS) containing 0.01% (v/v) Triton-X100 for 1 h, and reacted with an anti-HA antibody (1:500 dilution) at 4°C overnight followed by Texas Red conjugated with a goat anti-mouse IgG antibody (1:250 dilution). The mitochondria and the nucleus were stained with MitoTracker Green FM (1:500 dilution) and DAPI, respectively. Cell imaging was observed under a laser scanning microscopes (LSM 510, Carl Zeiss, Germany) equipped with 100x plan objective lens.
Time-laps monitoring for intracellular AMID mobilizations
HuH-7 cells were transfected with the expression vector for GFP alone or GFP-AMID. AMID mobilizations were monitored by detecting GFP signals with a laser scanning microscopy (LSM 510, Carl Zeiss, Germany) equipped with 100x plan objective lens every 5 min before and after application with adenosine (10 mM).
Separation into nuclear and cytosolic components
Cells were suspended in 400 μl of a cold buffer A [10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1% (v/v) protease inhibitor cocktail, pH 7.4] and lysed with 25 μl of 10% (v/v) Nonidet P-40 (Nacalai Tesque, Kyoto, Japan). After 15,000 × g centrifugation at 4°C for 5 min, the pellet was resuspended in 50 ul of a cold buffer B [20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1% (v/v) protease inhibitor cocktail, pH7.9] as a nuclear component and the supernatant was collected as a cytosolic component.
Samples were loaded on 10% (v/v) SDS-PAGE and transferred to polyvinylidene difluoride membrane. After blocking with TBST [20 mM Tris, 150 mM NaCl, 0.1% (v/v) Tween-20
] containing 5% (v/v) of bovine serum albumin, blotting membrane was reacted with an anti-AMID antibody (Santa Crutz Biotechnology, Santa Cruz, CA, USA) followed by an HRP-conjugated anti-goat IgG antibody. For β-actin detection, blotting membrane was reacted with an anti-β-actin antibody (SIGMA, Missouri, SL, USA) followed by an HRP-conjugated anti-mouse IgG antibody. Immunoreactivity was detected with an ECL kit (GE Healthcare, NJ, USA) and visualized using a chemiluminescence with an Image Gause software (FUJIFILM, Tokyo, Japan).
Cell cycle analysis
HuH-7 cells were transfected with the expression vector for GFP alone or GFP-AMID. Twenty-four hours later, cells were untreated and treated with adenosine (10 mM) for 24 h. Then, cells were harvested by a trypsinization, fixed with 70% (v/v) ethanol at 4°C overnight. Fixed cells were incubated in PBS containing 1.5 μg/ml RNase A for 1 h at 37°C, followed by staining with 5 μl of propidium iodide (PI) for 20 min on ice. Then, cells were collected on a nylon mesh filter (pore size, 40 μm), and cell cycles including the sub-G0 phase (apoptosis) were assayed in 1 × 104 GFP-positive cells with a flowcytometer (FACSCalibur, Becton Dickinson, USA) at an excitation of 488 nm and an emission of 585 nm, and analyzed using a Mod Fit LT software (Verity Software House Inc., Topshan, USA).
HuH-7 cells were transfected with the expression vector for HA alone or HA-AMID followed by G418 selection by the method as previously described [25
]. Twenty-four hours after transfection, cells were untreated and treated with adenosine (10 mM) for 24 h and then, harvested by adding trypsin. Cells were resuspended in a binding buffer and stained with both PI and annexin V-FITC, and loaded on a flow cytometer (FACSCalibur, Becton Dickinson, Franklin Lakes, USA) available for FL1 (annexin V) and FL2 (PI) bivariate analyisis. Data from 20,000 cells/sample were collected, and the quadrants were set according to the population of viable, unstained cells in untreated samples. CellQuest analysis of the data was used to calculate the percentage of the cells in the respective quadrants.
3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay
Cell viability was evaluated by a dye staining method using MTT as previously described [14
]. MTT-reactive cells were quantified at an absorbance of 570 nm using a micro-plate reader (SPECTRAmax PLUS384, Molecular Devices, Sunnyvale, USA), and percentage of independent basal levels (MTT intensities of cells untreated with any drug) was calculated.
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay
Cells were fixed with 4% (v/v) paraformaldehyde. After removing inactivate endogenous peroxidase with methanol containing 0.3% (v/v) H2O2, a Permeabilization Buffer was applied to cells and stood on ice for 5 min. Then, a Labeling Reaction Mixture was added and incubated in a humidified chamber at 37°C for 60 min. Reactive cells were stained with 3% (v/v) methyl green and detected with a light microscope.
Statistical analysis was carried out using unpaired t-test.