Figure 8

Effects of overexpressed dominant-negative IRF1 and overexpressed IRF8 on IFNγ-induced OPC apoptosis. A, OPCs were transfected by electroporation with the control vector (PCMV-IE-IRES-hrGFP-pA), the expression construct of a dominant-negative form of IRF1 (IRF1DN-hrGFP) which is a fusion protein of IRF1 DNA-binding domain (IRF1DBD) and hrGFP (PCMV-IE-IRF1DBD/hrGFP-pA), and the expression construct of rat IRF8 (PCMV-IE-IRF8-IRES-hrGFP-pA). Cells were cultured with GM for 24 h after transfection, and then treated with GM alone (control) or GM plus IFNγ (100 ng/ml). At 24 h after treatments, cells were stained with TMRE and DAPI, and analyzed by flow cytometry as in Figure. 7. B, Number of preapoptotic (TMRE^-/DAPI^-) OPCs in the cultures subjected to electroporation with the control vector or the IRF1DN expression construct at 24 h after treatment with IFNγ (100 ng/ml). Transfected (hrGFP⁺) and untransfected (hrGFP^-) populations were analyzed separately, using the same gatings as in Figure. 7. C, Number of live transfected cells (DAPI^-/hrGFP⁺) in the cultures transfected with the control vector, IRF1DN expression construct or IRF8 expression construct after a 24 h IFNγ-treatment (48 h after transfection). Percentages of DAPI^-/hrGFP⁺ cells in total live (DAPI^-) cells were calculated in each condition, and are shown as fold changes of the percentages just before addition of IFNγ (24 h after transfection). Note that the same percentages of transfected (hrGFP⁺) and untransfected (hrGFP^-) OPC populations died during 24 h after addition of IFNγ in the control group, whereas less and more transfected OPCs were dead in the IRF1-DN and IRF8 groups, respectively. ** Indicates p < 0.01 in comparison with the corresponding data at 24 h (n = 9).