FIG 5.

Effects of alterations in Jak-Stat pathway on PrPres formation. (A) Immunoblotting of PrPres in N167 cells treated with Jak inhibitor. Cells were treated with Jak inhibitor I at the indicated doses for 3 days. The amount of DMSO was equilibrated in all cell culture wells and was less than 0.2%. (B) Immunoblotting of PrPres and Stat proteins in N167 cells treated with siRNAs against Stat genes. Cells were treated for the last 3 days before analysis with the indicated doses of either a mixture of four siRNAs against Stat1 (si-Stat1 pool) or Stat2 (si-Stat2 pool) or each of three siRNAs against Stat2 (si-9, si-10, and si-11). The amount of transfection reagent was equilibrated in all cell culture wells. (C) mRNA levels of Stat genes in N167 cells treated with siRNAs against Stat genes. Cells were treated as described for panel B and were analyzed 24 or 48 h after transfection. Data are averages and standard deviations based on results of triplicate experiments (*, P < 0.01 versus control [transfection reagent alone] at each time point). (D) mRNA levels of the PrP gene in N167 cells treated with siRNAs against Stat genes. Cells were treated and analyzed as described for panel C. Data are averages and standard deviations based on results of triplicate experiments (**, P < 0.05 versus the control [transfection reagent alone] at each time point). (E) Immunoblotting of PrPres in N167 cells treated with siRNAs against IFN-stimulated genes. Cells were treated for the last 3 days before analysis with the indicated doses of either a mixture of four siRNAs (si-RNA pool), against Cxcl10, Ifit1, Ccl5, and Isg15, or each of three siRNAs against Oasl2 (si-1, si-2, and si-3). The amount of transfection reagent was equilibrated in all cell culture wells. (F) mRNA levels of IFN-stimulated genes in N167 cells treated with siRNAs against IFN-stimulated genes. Cells were treated as described for panel E and were analyzed 48 h after transfection. Data are averages and standard deviations based on results of triplicate experiments (*, P < 0.01 versus control [transfection reagent alone]). (G) Effect of Jak inhibitor on IFN-α-induced PrPres restoration in N167 cells. Cells were treated with 5 μg/ml Gly-9, 5 U/ml IFN-α, and 50 nM Jak inhibitor I (Jak inh) for 3 days. Each vehicle was used as a negative control. Graphic data are averages and standard deviations for triplicate immunoblot signals (**, P < 0.05; n.s., not significant). (H) Temporal profiles of mRNA levels of representative IFN-stimulated genes in Gly-9-treated N167 cells. The mRNA levels of Cxcl10 and Ifit1 were analyzed in N167 cells treated with 5 μg/ml Gly-9 or its vehicle for the indicated periods. Data are averages and standard deviations for triplicate experimental results (n.s., not significant; **, P < 0.05; *, P < 0.01).