Reagents
Polyinosinic polycytidylic acid (poly-IC), and lipopolysaccharide (LPS) from Escherichia coli, and an actin antibody were obtained from Sigma (St Louis, MO, USA). The TLR7 ligand R848 and TLR9 ligand CpG were purchased from InVivoGen (San Diego, CA, USA) and Novus Biologicals (Centennial, CO, USA), respectively. The Illustra RNAspin kit was obtained from GE Healthcare (Buckinghamshire, UK). Rabbit antibodies against ISG20 and IFN-inducible protein with tetratricopeptide repeat 1 (IFIT1) were purchased from GeneTex (Irvine, CA, USA). Lipofectamine RNAiMAX reagent, Moloney murine leukemia virus (MMLV) reverse transcriptase, dNTP mix, and small interfering RNA (siRNA) against IFN-β were obtained from Thermo Fisher Scientific (Waltham, MA, USA). siRNA against ISG20 and a nontargeting negative control siRNA were obtained from Qiagen (Hilden, Germany). The SsoAdvanced Universal SYBR Green Supermix was obtained from Bio-Rad (Hercules, CA, USA). Horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies were obtained from Medical & Biological Laboratories (Nagoya, Japan). Polyvinylidene difluoride (PVDF) membranes and Luminata Crescendo Western HRP substrate were obtained from Merk Millpore (Darmstadt, Germany). Enzyme-linked immunosorbent assay (ELISA) kit for CX3CL1 was obtained from R&D Systems (Minneapolis, MN, USA).
Cells
Normal human GECs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). Cells were cultured in endothelial growth medium-2 (EGM-2, Lonza, Walkersville, MD, USA) on gelatin-coated plates, as previously described [18-20]. Cells were treated with 30 μg/mL poly-IC, 1 μg/mL LPS, 5 μg/mL R848, or 100 μg/mL CpG for up to 24 h. To examine the concentration-dependent effects of poly IC, cells were treated with 0.5-50 μg/mL poly-IC for 16 h. For siRNA treatment, cells were cultured in a medium without antibiotics for 24 h and then transfected with specific siRNA against IFN-β, ISG20, or a nontargeting negative control siRNA using the Lipofectamine RNAiMAX reagent according to the supplier’s protocol. After 48 h of incubation, cells were treated with 30 µg/mL poly-IC.
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells using the Illustra RNAspin kit. Single-stranded complementary DNA (cDNA) was synthesized from total RNA using oligo(dT)18 and MMLV reverse transcriptase. ISG20, CX3CL1, and CXCL10 expression was quantified using specific primers and SsoAdvanced Universal SYBR Green Supermix. 18S ribosomal RNA (18SrRNA) was used as an internal control.
The sequences of the primers used are as follows:
ISG20-F: 5’- ATCTCTGAGGGTCCCCAAGGA -3’,
ISG20-R: 5’- TTCAGTCTGACACAGCCAGGCG -3’,
CX3CL1-F: 5’- GACCCCTAAGGCTGAGGAAC -3’,
CX3CL1-R: 5’- CTCTCCTGCCATCTTTCGAG -3’,
CXCL10-F: 5’- TTCAAGGAGTACCTCTCTCTAG -3’,
CXCL10-R: 5’- CTGGATTCAGACATCTCTTCTC -3’,
18S-F: 5’- ACTCAACACGGGAAACCTCA -3’,
18S-R: 5’- AACCAGACAAATCGCTCCAC -3’.
Western blotting
After incubation, cells were lysed using the Laemmli reducing sample buffer. Lysates were subjected to 5-20% polyacrylamide gel electrophoresis. The separated proteins were transferred to a PVDF membrane, which was subsequently blocked with nonfat dry milk for 2 h at room temperature and incubated with antibodies against ISG20 (1:1,000), IFIT1 (1:5000), or actin (1:5000) for 18 h at 4℃. After washing, the membranes were incubated with the HRP-conjugated anti-rabbit IgG antibody. The Immobilon Crescendo Western HRP chemiluminescence substrate was used for detection.
ELISA
The concentration of CX3CL1 protein in the cell-conditioned medium was measured using a commercially available ELISA kit, according to the manufacture’s recommended protocol.
Statistical analysis
Data are presented as the mean ± standard deviation (SD). The Mann-Whitney U test was used to assess significant differences. Statistical significance was set at p < 0.05.