Mifn β

HEK293 cells were grown in DMEM supplemented with 10% FBS and transfected with Fugene-HD Transfection Reagent (Roche), reporter constructs (pAhR-Luc/pTK-Renilla or pGud-Luc/pTK Renilla) as well transcription factor expression constructs (pStat1, pIrf9) as indicated. In some experiments, transfected cells were exposed to 500 IU/ml of mIFN-β (Bio X cell) or AhR inhibitor CH223191 (Sigma). For the measurement of AhR ligands in human serum, 15.000 HEK293 cells were plated in 96 well plates 24 h before transfection with pGud-Luc and pTK-Renilla. After 24 h transfected cells were incubated with DMEM supplemented with 10% of patient serum. Luciferase activity was analyzed 24 h later using Dual Luciferase Reporter System (Promega) and normalized to Renilla luciferase activity.

Ifnar1 expression was knocked down in mouse primary astrocytes in culture using a lentivirus vector carrying an Ifnar1-targeting shRNA (TRCN0000374694), a lentivirus carrying a non-targeting sequence was used as a control (TRCN0000018782) (Sigma). Astrocytes were incubated with lentiviruses and 8 μg/ml polybrene (both from Sigma-Aldrich) for 24 h and thereafter incubated with mIFN-β (Bio-X-cell) or vehicle. GFP⁺ astrocytes were sorted, and gene knockdown was verified by qPCR. Transduction efficiency as determined by GFP-expression in transduced astrocytes was around 20% (Fig. 2b and not shown).