Rate of PrPres depletion, synthesis and clearance in the presence or absence of swa in PK1 cells infected with swa-sensitive or swa-dependent RML prions.

aPK1 cells were infected with brain-derived, swa-sensitive RML prions or swa-dependent prions (RML(AMO10+swa)/(PK1+swa)14, derived from RML propagated sequentially in AMO10 cells and PK1 cells in the presence of swa; similar to in Figure 2B, however passaged in PK1 +swa for 14 rather than 20 splits). Cells were propagated for 8 days and seeded into two 15-cm dishes, to one of which 2 µg swa/ml were added. Three days after swa addition, cells were transferred to ten 15-cm dishes, resulting in 10 dishes with and 10 without swa. One day after the third split, two dishes were harvested (time zero) and 1 unit PIPLC/ml media was added to four dishes of each condition, following which one dish per condition was harvested after 7,12,18 and 24 hours. For each sample, cells were counted and PrPres levels were determined by western blot analysis (see Figure 9). kde = PrPres depletion rate; ks and kcl are the rates of PrPres synthesis and clearance, respectively; kp is the rate of cell partitioning = negative of cell growth rate, and includes data points from 0 to 24 hours. kde, ks and kcl are calculated from the 7- to 24-hour points by the equation kde = ks+kcl+kp, with ks = 0 in the presence of PIPLC under the reasonable assumption that kcl is the same in the presence or absence of PIPLC. The experiment was evaluated from triplicate western blots. The Table shows that in PK1 cells the clearance rate of swa-dependent PrPres was 3× lower in absence of swa, and 5.5× lower in the presence of swa than that of swa-sensitive RML (t-test evaluation:*p = 0.015 and**p = 0.0025, respectively).