Among hundreds of ISGs, some members of the IRF protein family are immediate transcriptional targets mainly of inter feron mediated JAK STAT signaling, and subsequently control induction of downstream ISGs as transcription regulators. Indeed, we previously demonstrated that IRF1 Inhibitors,Modulators,Libraries and IRF9 transcriptional kinetics differ between IFNg treated and IFNb treated OPCs. IFNg elicited a more than 70 fold sustained elevation of IRF1 mRNA from the basal levels in OPCs. In contrast, IFNb mediated up regulation of IRF1 mRNA was transi ent even in the continuous presence of IFNb, falling to less than one tenth of the sustained levels induced by IFNg at 24 h. We extended this analysis to other mem bers of the IRF protein family to obtain a comprehensive view of differential transcriptional regulation of all known IRFs in response to IFNg and IFNb, because at least some members are able to heterodimerize.

The quantitative PCR results demonstrated that members of the IRF protein family in OPCs could be classified into three groups in terms of their distinctive Inhibitors,Modulators,Libraries patterns of tran scriptional induction by IFNg and IFNb, 1 IRF1 and IRF8 were preferentially up regulated by IFNg compared with IFNb, 2 IRF7 was preferentially up regulated by IFNb compared with IFNg, and 3 IRF2 to IRF6 and IRF9 were similarly regulated or not regulated by IFNg and IFNb, with the basal levels of the transcripts being IRF2 IRF3 IRF9 IRF6 IRF5. IRF4 mRNA was below the detection limit in OPCs even in the presence of IFNs.

We therefore focused on roles for Inhibitors,Modulators,Libraries IRF1 and IRF8 in IFNg induced apoptosis of OPCs in this study, because IRF1 mRNA and IRF8 mRNA were up regulated within 1 hr after addition of IFNg, and remained at more than 10 fold Inhibitors,Modulators,Libraries higher levels than those induced by IFNb until at least 24 h. Immunoblotting for IRF1 and IRF8 pro teins also confirmed selective up regulation of these pro teins in the IFNg treated OPC cultures. IRF1 mediates IFNg induced OPC apoptosis We examined the effects Inhibitors,Modulators,Libraries of forced expression of either IRF1 or IRF8 on OPC viability. Since transient transfec tion of primary rat OPCs generally demonstrates limited efficiency, we used the dual expression constructs PCMV IE IRF1 IRES hrGFP pA and PCMV IE IRF8 IRES hrGFP pA in order to discriminate transfected cells from untransfected cells with the aid of coexpressed hrGFP in the transfected cells. PCMV IE IRES hrGFP pA was employed as control.

These dual expression constructs and the conventional cell death assay depending on the membrane impermeable DNA binding dye DAPI enabled us to count preapoptotic cells in either hrGFP or hrGFP population by flow cytometry with the gating strategy Temsirolimus mTOR shown in Figure. 7B. Overexpres sion of IRF1 significantly increased the number of prea poptotic cells in the transfected population at 6 and 24 h after transfection.