Figure 2.

Loss of PrP^C exacerbates cell sensitivity to sTNFα by reducing TNFR1 shedding in a ROCK-I- and PDK1-dependent manner. (a) Reduced viability of PrP^null-1C11 cells after exposure to increasing sTNFα concentrations for 72 h as compared to 1C11 cells (CTRL). (b) Increased dendritic fragmentation in PrP^0/0-CGNs after exposure to increasing concentrations of sTNFα for 72 h compared to wild-type CGNs (WT). For figures a and b, LD₅₀ ^TNFα values are indicated in Table 1. (c) Immunofluorescence experiments showing enhanced level of TNFR1 at the cell surface of PrP^null-1C11 and PrP^null-1C11^5-HT cells as well as PrP^0/0 CGNs as compared to their corresponding PrP^C expressing cells (CTRL, WT). Scale bar = 50 µm. (d) ELISA-based quantification experiments indicating reduced concentration of sTNFR1 in the culture medium of PrP^null-1C11/1C11^5-HT cells compared to PrP^C expressing cells. *p < 0.01. (e) Western blots showing a stronger activation of caspase-3 in PrP^null-1C11^5-HT cells exposed to sTNFα (10 ng ml⁻¹) for 120 min than in PrP^C expressing cells. Antagonizing either ROCK activity with Y-27632 (100 µM for 1 h) or PDK1 activity with BX912 (1 µM for 1 h) in PrP^C-depleted cells reduces toxic action of sTNFα. ^# p < 0.05 vs. PrP^C-expressing cells exposed to sTNFα. ^## p < 0.05 vs. PrP^null-cells treated with sTNFα. Data shown are the mean ± SEM from three experiments performed in triplicate.