Gaussia and cypridina luciferase assay kit

The 293T cells were co-transfected with pGLuc-5′-UTR or pGLuc-ASP, together with pCMV-CLuc (New England BioLabs, Ipswich, MA, USA) using Lipofectamine 2000 reagent. At 24 h after transfection, cells were treated with the indicated concentrations of test compounds. After 3 days of treatment, the Gaussia and Cypridina luciferase activities were measured using the Gaussia and Cypridina Luciferase Assay Kit (New England BioLabs) according to the manufacturer’s instructions. Gaussia luciferase activity was normalized to the corresponding Cypridina luciferase activity.

For the MLT1A0 promoter assay, 1 × 10⁵ 293T cells were seeded in 24-well plates. After the one-day incubation, the cells were transfected with 0.2 µg of pGLuc-Basic or pGLuc-MLT1A0 together with 10 ng of pCMV-CLuc (New England BioLabs) using Lipofectamine 2000 (Invitrogen, Gaithersburg, MD, USA). At 24 h after the transfection, the cells were further transfected with 0.4 µg of polyI:C (Sigma-Aldrich, St. Louis, MO, USA). At 48 h after reporter transfection, the luciferase activity of the cells was measured using the Gaussia and Cypridina Luciferase Assay Kit (New England BioLabs) according to the manufacturer’s instructions. For the IFN promoter assay, 293T cells were transfected with 0.2 µg of p125-Luc and 10 ng of pRL-TK (Promega, Fitchburg, WI, USA) with or without 0.2 µg of pCMV-MLT-int. At 24 h after reporter transfection, the cells were further transfected with 0.4 µg of polyI:C. For the ISRE reporter assay, 293T cells were transfected with 0.2 µg of pISRE-Luc (Agilent Technology, Wilmington, DE, USA) and 10 ng of pRL-TK with or without 0.2 µg of pCMV-MLT-int. At 24 h after reporter transfection, the cells were further treated with 1 KU of universal IFN (Sigma-Aldrich). At 48 h after reporter transfection, the luciferase activity of the cells was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.