FIGURE 6.

Coexpression of wild type PrP in SH/Sho cells eliminates both Zeocin and G418 hypersensitivity caused by Shadoo expression. A, SH/Sho+GFP or SH/Sho+PrP cells have slightly higher Shadoo expression than the parental SH/Sho cells. Shown is Western blotting analysis of extracts from various cells as indicated above the lanes. Samples were treated with PNGase F. B, PrP expression is detectable only in SH/Sho+PrP cells. Western blotting analysis of extracts from the same cells as in A. Endogenous PrP is below the detection limit. A and B, *, a nonspecific band. Numbers and marks on the left indicate the positions of the corresponding molecular size markers in kDa. β-Actin was used as a loading control. C–F, cytotoxicity assays, using PrestoBlue reagent. C and D, coexpression of PrP but not EGFP diminishes Zeocin hypersensitivity caused by Shadoo expression (48-h treatment). C, representative experiment carried out at Zeocin concentrations between 0 and 100 μg/ml on various cells as indicated. D, bars show the means ± S.D. (error bars) of cell viabilities measured at 6.25 μg/ml Zeocin concentration in n = 4 independent experiments. E and F, PrP coexpression diminishes G418 hypersensitivity caused by Shadoo expression (48-h treatment). E, representative experiment carried out at G418 concentrations between 0 and 1000 μg/ml on various cells, as indicated. F, bars show the means ± S.D. of cell viabilities measured at 250 μg/ml G418 concentration in n = 3 independent experiments. C–F, 100% is the fluorescence value of untreated controls of each cell line. C and E, values are means ± S.D. of corresponding replicas within the experiment. D and F, samples were compared with SH/Sho cells; *, p < 0.05; **, p < 0.01; ***, p < 0.001.