FIGURE 7.

Shadoo expression causes Zeocin and G418 hypersensitivity in HEK293 cells that is eliminated by the coexpression of wild type PrP. A, HEK/Sho+GFP or HEK/Sho+PrP cells have slightly higher Shadoo expression than the parental HEK/Sho cells. Shown is Western blotting analysis of extracts from various cells as indicated above the lanes. *, a nonspecific band. B, PrP expression is detectable only in HEK/Sho+PrP cells. Shown is Western blotting analysis of extracts from the same cells as in A after PNGase F treatment. A and B, 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, Shadoo expression causes Zeocin hypersensitivity in HEK cells, which is diminished by the coexpression of PrP but not EGFP (48-h treatment). C, average of three independent experiments carried out at Zeocin concentrations between 0 and 200 μg/ml on various cells as indicated. D, bars show the means ± S.D. of cell viabilities measured at 50 μg/ml Zeocin concentration in n = 3 independent experiments. E and F, Shadoo expression causes G418 hypersensitivity in HEK cells that is diminished by the coexpression of PrP but not EGFP (48-h treatment). E, average of three independent experiments carried out at G418 concentrations between 0 and 1000 μg/ml on various cells as indicated. F, bars show the means ± S.D. (error bars) 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 three independent experiments. D and F, viabilities were compared using a Tukey post hoc test after one-way analysis of variance; asterisks above bars, difference when compared with HEK/Sho; brackets, significant difference between compared cells; *, p < 0.05; ***, p < 0.001.