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. 2014 Apr;88(8):4083–4099. doi: 10.1128/JVI.03775-13

FIG 7.

FIG 7

Pde4dip involvement in PrPc and PrPres. (A) Temporal profile of Pde4dip mRNA levels in Gly-9-treated N167 cells. Cells were 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). (B) Immunoblotting of PrPres in N167 cells and PrPc in N2a cells treated with Gly-9 and Pde4dip gene knockdown. Cells were treated with 5 μg/ml Gly-9 or its vehicle (DMSO) for three and a half days and, simultaneously, with the indicated doses of siRNA against Pde4dip (si-Pde4dip; called si-5 in Fig. 6C) for the last 3 days before harvest. Graphic data are averages and standard deviations for triplicate immunoblot signals (n.s., not significant; *, P < 0.01; **, P < 0.05). (C) mRNA levels of Pde4dip and PrP in N167 cells treated with Gly-9 and si-Pde4dip. Cells were treated as described for panel B, and si-Pde4dip was used at a dose of 10 nM. The vehicle for Gly-9 and the transfection reagent for si-Pde4dip were used as negative controls. Data are averages and standard deviations for triplicate experimental results (*, P < 0.01; n.s., not significant). (D) Immunofluorescence of abnormal PrP accumulation in N167 cells treated with Gly-9 and si-Pde4dip. Cells were treated as described for panel C. Nuclei were stained with Hoechst 33258 (H33258). (E) Flow cytometry of cell surface PrP and lipid rafts in N2a cells treated with Gly-9 and si-Pde4dip. Cells in the presence of 5 μg/ml Gly-9 or vehicle (DMSO) were treated with transfection reagent alone (cont) or 5 nM si-Pde4dip (siRNA) for 3 days as described for panel B. Cell surface PrP (anti-PrP) and lipid rafts (cholera-toxin B) were labeled as described earlier in this report. (F) Immunoblotting of autophagosome-related LC3-II in N167 cells treated with Gly-9 and si-Pde4dip. Cells were treated as described for panel C. Treatment with 100 mM trehalose is shown as a positive control. Graphic data are averages and standard deviations for triplicate immunoblot signals (n.s., not significant).