ISRE-Luciferase Assay for STAT1 Signaling

Luciferase assay was performed in 96-well plate using the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer's instructions. In brief, adherent HEK293 cells were transiently transfected (lipofectamine, Invivogen) with the ISRE (interferon sensitive responder element) luciferase responder plasmid [66 (link)] and the Renilla plasmid used for normalization. For experiments involving STAT1 silencing, siSTAT1 or siNT were transiently transfected using interferin reagent (Polyplus) 24 h before reporter plasmid transfection. Twenty four hours later, cells were stimulated for 24 h with CM^veh or CM^mafo, containing or not AG490 (50 μM) or DMSO (vehicle). The luminescence activity in cell lysates was measured using a Mithras LB940 (Berthold) and data were analyzed using the MicroWin2000 software.