Figure 7
Overexpressed IRF1 induces OPC apoptosis, whereas overexpressed IRF8 does not. A, OPCs were transfected with the control vector (PCMV-IE-IRES-hrGFP-pA), the expression construct of rat IRF1 (PCMV-IE-IRF1-IRES-hrGFP-pA), or the expression construct of rat IRF8 (PCMV-IE-IRF8-IRES-hrGFP-pA) by electroporation. These OPCs were incubated with TMRE and DAPI at 6 and 24 h after transfection and analyzed by flow cytometry. B, Representative gating scheme of flow cytometric analysis of preapoptotic (TMRE-/DAPI-) OPCs in the transfected (hrGFP+) and untransfected (hrGFP-) populations at 24 h after transfection with the IRF1 expression construct. Live cells negative for DAPI in the R2 gate were separated into hrGFP + (R3) and hrGFP - (R4) cells. Preapoptotic OPCs were then counted in the R5 and R6 gated areas in hrGFP + (R3) and hrGFP - (R4) populations, respectively. C, Number of preapoptotic (TMRE-/DAPI-) OPCs in transfected (hrGFP+) and untransfected (hrGFP-) populations at 6 and 24 h after transfection with the control vector, IRF1 expression construct or IRF8 expression construct. D, Reduction in live transfected cells (DAPI-/hrGFP+) at 24 h in the cultures transfected with the control vector, IRF1 expression construct or IRF8 expression construct. Due to the different transfection efficiencies among the expression constructs, percentages of live transfected (DAPI-/hrGFP+) cells in total live (DAPI-) cells were calculated in each condition, and are shown as fold changes of the calculated percentages at 6 h after transfection. ** Indicates p < 0.01 (n = 9). E, Overexpressed IRF1 and IRF8 were verified by immunoblotting. GFP-positive cell populations in OPC cultures transfected with the expression constructs of rat IRF1 (IRF1-GFP+) or IRF8 (IRF8-GFP+) were separated from GFP-negative cell populations (IRF1-GFP- and IRF8-GFP-) with a cell sorter, and subjected to immunoblotting for IRF1 and IRF8, respectively. Untransfected OPCs cultured with medium alone (C), and medium containing IFNγ (100 ng/ml, G) were used as negative and positive controls, respectively.