Reagents
Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The poly(I:C)-low molecular weight (LMW)/LyoVec™ and poly(I:C)-high molecular weight (HMW)/LyoVec™ were purchased from InvivoGen (San Diego, CA, USA). Rabbit anti-human RIG-I (cat no. 4520), MDA5 (cat no. 5321) monoclonal antibodies and anti-rabbit immunoglobulin G (IgG) horseradish peroxidase (HRP)-linked (cat no. 7074) antibody were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). Goat anti-human actin polyclonal antibody (sc-1615) and HRP-conjugated donkey anti-goat IgG (sc-2056) were purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA).
Cell culture
THP1 human acute monocytic leukemia cells were obtained from RIKEN BioResource Center (Tsukuba, Japan). Cells were cultured in RPMI-1640 supplemented with 1% penicillin and streptomycin (Gibco, Grand Island, NY, USA) and 10% heat-inactivated fetal bovine serum (Japan Bioserum Co., Ltd., Fukuyama, Japan) at 37°C in a humidified atmosphere containing 5% CO2. THP1-derived macrophages (macrophage-like cells) were prepared as previously described (8). THP1 cells (2.0×105 cells/ml) were plated in 60-mm dishes (Iwaki, Tokyo, Japan) with 4 ml of medium containing 100 ng/ml PMA and cultured for 48 h. After the 48-h culture, the medium containing PMA was replaced with fresh medium not containing PMA and macrophage-like cells were used in experiments.
In vitro X-irradiation
X-irradiation (150 kVp, 20 mA, 0.5 mm Al and 0.3 mm Cu filters) was performed using an X-ray generator (MBR-1520R-3; Hitachi Medical Corporation, Tokyo, Japan) at a distance of 45 cm from the focus and a dose rate of 1.00 Gy/min.
Stimulation with poly(I:C)/LyoVec™
To stimulate RLRs, two types of poly(I:C)/LyoVec™ (InvivoGen), which is a complex of poly(I:C) and the transfection reagent LyoVec™, were used. In brief, macrophage-like cells were exposed to X-rays and 500 ng/ml poly(I:C)-LMW/LyoVec™ or poly(I:C)-HMW/LyoVec™ was added to the culture 24 h after X-irradiation. After an additional 24 h, cells were harvested for reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis.
RT-PCR
Total RNA was extracted using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) and quantified using a NanoDrop (Thermo, Wilmington, DE, USA). cDNA templates were synthesized from 1 µg RNA using the iScript cDNA Synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), according to the manufacturer's instructions. PCR was performed using the AccuPrime™ Taq DNA Polymerase system (Invitrogen Life Technologies, Carlsbad, CA, USA). The primer sequences used are shown in Table I. The reaction conditions for RIG-I were 94°C for 1 min followed by 30 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, and subsequently 72°C for 10 min. The reaction conditions for MDA5 were 94°C for 1 min followed by 30 cycles of 94°C for 1 min, 64°C for 1 min and 72°C for 1 min, and subsequently 72°C for 10 min. The reaction conditions for IFN-β and β-actin were as reported elsewhere (8). The PCR products were confirmed using electrophoresis on ethidium bromide-stained 1.5% agarose gels.
SDS-PAGE and western blotting
Cells were harvested and suspended in CelLytic™ M Cell Lysis reagent (Sigma-Aldrich) containing 1% Protease Inhibitor cocktail (Sigma-Aldrich) on ice for 30 min. After centrifugation at 20,600 x g for 20 min at 4°C, supernatants were collected. The protein concentration was determined using the Bio-Rad Protein Assay kit and a SmartSpec™ plus spectrophotometer (Bio-Rad Laboratories, Inc.). Each lysate was mixed with 2X sample buffer (Bio-Rad Laboratories, Inc.) containing 5% 2-mercaptoethanol. After boiling for 5 min, proteins were separated using 4–20% Mini-PROTEAN® TGX™ Precast gels (Bio-Rad Laboratories, Inc.) and transferred onto polyvinylidene difluoride membranes of Trans-Blot® Turbo™ Mini PVDF Transfer pack (Bio-Rad Laboratories, Inc.) using the Trans-Blot® Turbo™ Transfer system (Bio-Rad Laboratories, Inc.). The membranes were blocked in TBST buffer [10 mmol/l HCl (pH 7.5), 100 mmol/l NaCl and 0.1% Tween-20] containing 4% ECL Prime Blocking agent (GE Healthcare UK Ltd., Little Chalfont, England). The membranes were probed with each primary antibody in Can Get Signal® Immunoreaction Enhancer solution 1 (Toyobo, Co., Ltd, Osaka, Japan) overnight at 4°C. Following the reaction with the primary antibodies, the membranes were labeled with HRP-conjugated secondary antibodies in Can Get Signal® Immunoreaction Enhancer solution 2 (Toyobo, Co., Ltd) for 1 h. The antigens were visualized by the ECL Prime western blotting detection system (GE Healthcare UK Ltd.).