Figure 11. Effect of lysosomal hydrolysis inhibition on the decrease of PrP^Sc levels induced by CPZ or U18666A treatment.

(A) Immunoblot analysis of PrP-res and cathepsin D. ScN2a-3-22L cells were cultured with 10 µM CPZ or 5 µM U18666A or without compounds for 24 h. Subsequently, monensin (Mon) or bafilomycin A1 (BafA1) was added to the culture at a final concentration of 100 or 5 nM, respectively. Following an additional incubation for 36 h with or without Mon or BafA1, the cells were subjected to immunoblotting for PrP-res, cathepsin D, or β-actin. Representative immunoblot images are shown on the left. The bracket in the immunoblot of cathepsin D denotes the pro- and/or intermediate forms of cathepsin D (Pro/Int). The arrowhead denotes the mature form of cathepsin D (M). The upper right graph shows the levels of PrP-res relative to the control. The lower right graph shows the ratio of mature to pro−/intermediate forms of cathepsin D. The means and SDs of three independent experiments are depicted. Asterisks indicate a significant difference between Mon- or BafA1-treated samples and untreated samples (non-treated) (Student’s t-test, p<0.05). (B) Localization of PrP^Sc. ScN2a-3-22L cells grown on a chambered coverglass were cultured with 10 µM CPZ for 24 h. Subsequently, Mon or BafA1 was added at a final concentration of 100 or 5 nM, respectively, and the cells were cultured for additional 24 h in the presence of CPZ and Mon or BafA1. The cells were subjected to double-staining of PrP^Sc and Lamp1 before (left) and after the treatment with Mon (middle) or BafA1 (right). The upper panel shows the merged images of PrP^Sc (green) and nuclei (blue). The bottom panel shows the merged images of PrP^Sc (green), Lamp1 (red) and nuclei (blue). Scale bars: 10 µm.