Fig 2. KDC203 treatment lowers PrPC levels in guinea pig cells and PrPC expression temporarily overshoots when KDC203 is withdrawn.
(A) The guinea pig cell line 104C1 which exhibits a fibroblast morphology responds to 7-day treatment with KDC203, but not Ouabain, with a significant reduction in its PrPC levels. However, the KDC203 concentrations required for this outcome exceed approximately 30-fold those observed to achieve a similar PrPC reduction in human cells. The asterisk indicates high molecular weight ATP1A1 antibody-reactive bands in a subset of fractions. Although the identity of these bands has not been resolved, their presence is consistent with partially SDS-stable ATP1A1-containing complexes. (B) The epithelial guinea pig cell line, GPC-16, resembles 104C1 cells in their response to KDC203 or Ouabain. A Coomassie stain of the western blot membrane validated equal protein loading. ATP1A1 signals were revealed following a reprobe of the PrPC blot. (C) Guinea pig 104C1 cells recapitulate reduced PrPC lowering potency of KDC203 when grown in media with higher serum levels. Note that the relatively stable ATP1A1 levels serve partially as a loading control as they were revealed on the same membrane following a reprobe of the PrPC blot. (D) Upon PI-PLC cleavage of guinea pig 104C1 cells, no PrPC can be detected in cellular lysates, and the PI-PLC releasable pool is diminished in KDC203 treated cells. (E) By its apparent molecular weight, PrPC expressed in guinea pig 104C1 cells is predominantly composed of the N-terminally truncated C2 isoform. (F) Daily KDC203 treatment of guinea pig 104C1 cells caused a time-dependent reduction of steady-state PrPC levels, culminating in a profound reduction after 7 days of treatment. The subsequent withdrawal of KDC203 reveals a temporary upregulation of the underlying PrPC expression whose magnitude correlates with the prior KDC203 concentration used to achieve the PrPC lowering effect. This effect ceases after 3 days of KDC203 withdrawal.