We used two different ISRE-luciferase reporter assays: (i) an assay using the ISRE3 reporter plasmid (pGL4.10[luc2] backbone, Promega #E6651), which contains, as previously described158 (link), three repeats of the ISRE sequence (5’-GGAAAGGGAAACCGAAACTGAA-3’) separated by spacers designed on the basis of the ISRE motif from the ISG15 promoter, (ii) an assay using the ISRE5 reporter plasmid, which contains, as previously described139 (link), five repeats of the ISRE sequence 5’-GGGAAAGTGAAACTA-3’. HEK293T cells were transiently transfected, in 96-well plates, with the (ISRE) reporter plasmid (100 ng/well and 100 μL DMEM-10% FBS medium), the pRL-SV40 vector (Promega, #E2231, 40 ng/well) and the IRF1 WT or mutant p.CMV6 plasmid (100 ng/well), with the Lipofectamine LTX kit (Thermo Fisher Scientific, #15338–100), according to the manufacturer’s instructions. Cells were used for the ISRE assay with the Dual-Luciferase system kit (Promega #E1980), according to the manufacturer’s protocol, 24 hours after transfection. Signal intensity was determined with a Victor X4 plate reader (Perkin Elmer). Experiments were performed in triplicate, and dual reporter activity is expressed as the fold-induction relative to cells transfected with the empty vector.
Rosain J., Neehus A.L., Manry J., Yang R., Le Pen J., Daher W., Liu Z., Chan Y.H., Tahuil N., Türel Ö., Bourgey M., Ogishi M., Doisne J.M., Izquierdo H.M., Shirasaki T., Le Voyer T., Guérin A., Bastard P., Moncada-Velez M., Han J.E., Khan T., Rapaport F., Hong S.H., Cheung A., Haake K., Mindt B.C., Perez L., Philippot Q., Lee D., Zhang P., Rinchai D., Al Ali F., Ata M.M., Rahman M., Peel J.N., Heissel S., Molina H., Kendir-Demirkol Y., Bailey R., Zhao S., Bohlen J., Mancini M., Seeleuthner Y., Roelens M., Lorenzo L., Soudée C., Paz M.E., Gonzalez M.L., Jeljeli M., Soulier J., Romana S., L’Honneur A.S., Materna M., Martínez-Barricarte R., Pochon M., Oleaga-Quintas C., Michev A., Migaud M., Lévy R., Alyanakian M.A., Rozenberg F., Croft C.A., Vogt G., Emile J.F., Kremer L., Ma C.S., Fritz J.H., Lemon S.M., Spaan A.N., Manel N., Abel L., MacDonald M.R., Boisson-Dupuis S., Marr N., Tangye S.G., Di Santo J.P., Zhang Q., Zhang S.Y., Rice C.M., Béziat V., Lachmann N., Langlais D., Casanova J.L., Gros P, & Bustamante J. (2023). Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria. Cell, 186(3), 621-645.e33.
Corresponding Organization : Howard Hughes Medical Institute
Other organizations :
Medizinische Hochschule Hannover, Institut de Recherche en Infectiologie de Montpellier, Centre National de la Recherche Scientifique, Université de Montpellier, Bezmiâlem Vakıf Üniversitesi, Ontario Genomics, McGill University, Institut Pasteur, Université Paris Sciences et Lettres, Institut Curie, University of North Carolina at Chapel Hill, St Vincent's Clinic, Garvan Institute of Medical Research, Garrahan Hospital, Ümraniye Eğitim ve Araştırma Hastanesi, Centro Científico Tecnológico - Tucumán, Hôpital Cochin, Hôpital Robert-Debré, Hôpital Saint-Louis, Department of Virology, Vanderbilt University Medical Center, Hôpital Necker-Enfants Malades, European Genomic Institute for Diabetes, Hôpital Ambroise-Paré, University Medical Center Utrecht, Utrecht University, Hamad bin Khalifa University, German Center for Lung Research
Transfection of HEK293T cells with IRF1 WT or mutant p.CMV6 plasmid (100 ng/well)
dependent variables
ISRE reporter activity measured using the Dual-Luciferase system kit
control variables
Transfection of HEK293T cells with the ISRE reporter plasmid (100 ng/well)
Transfection of HEK293T cells with the pRL-SV40 vector (40 ng/well)
Use of 96-well plates
Use of DMEM-10% FBS medium
Use of Lipofectamine LTX kit for transfection
Measurement of signal intensity with a Victor X4 plate reader
Experiments performed in triplicate
controls
Positive control: Cells transfected with the IRF1 WT plasmid
Negative control: Cells transfected with the empty vector
Annotations
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