Figure 7.

S3-PrP formed clusters under Opti-MEM culture conditions. RK13 cells expressing WT-PrP (WT), S3-PrP (S3), and F88W-PrP (F88W) were cultured in Opti-MEM or DMEM. A, the cells were harvested at 10 days of culture, and the expression levels of PrP were determined by Western blot analysis. Amounts of PrP immunoreactive species >24 kDa and <24 kDa are presented (also see Fig. S7). B, upper panel, representative images of each cell line grown in Opti-MEM (top and middle). Lower panel, frequency of clustered PrP particles with size ranging from 1 to 30 μm² (bottom). PrP in red; β-tubulin in green; and nuclei in blue. The scale bars represent 10 μm. C, degree of clustered PrP was quantified by the coefficient of variation (CV) from regions exhibiting above threshold PrP signals. N = 60, 62, and 87 for WT, S3, and F88W, respectively. A total of 24 different sites (160 μm² for each) were analyzed. Data were presented as percent frequency of the CVs. D, mean comparisons of CVs from each cell line cultured in Opti-MEM (left, the same data shown in C) and DMEM (right, n = 60, 82, and 85 for WT, S3, and F88W, respectively). A total of 18 different sites, 160 μm² for each, were analyzed). E, integrated PrP signal densities (IntDen). F, sizes of individual cells (area) were analyzed from a total of 594 cells grown in DMEM and Opti-MEM. N = 154, 194, and 246 for WT, S3, and F88W, respectively. Error bars represent SD. ∗p < 0.05 and ∗∗p < 0.01 by one-way ANOVA with Tukey’s multiple comparison test (A, D, E, and F) and unpaired t test (C). The equality of variance was analyzed by F statistics (B). CV, coefficient of variation; DMEM, Dulbecco's modified Eagle's medium; PrP, prion protein.