Summary of the standard scrapie cell assay and variants developed in this study.

<p>Summary of the standard scrapie cell assay and variants developed in this study.</p

Schematic description of dendrimers PPIg4, PPIg5 and mPPIg5 used in this study.

<p><b>A</b>) mPPIg5 is obtained by reductive amination of PPIg5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055282#pone.0055282-Klajnert1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055282#pone.0055282-Fischer1" target="_blank">[14]</a>. <b>B</b>) Molecular structure of cationic 4th generation poly(propylene imine) (PPIg4) with the smallest size used in biological experiments. <b>C</b>) Molecular weights for the dendrimers utilised in this study.</p

mPPIg5 treatment of ScN2a cells and treatment of lysates from RML and 22L infected N2a cells.

<p><b>A</b>) RML infected N2a cells and 22L infected N2a#58 cells were lysed in 4% sarkosyl (in PBS pH 7.4). 100 µg of lysate was then left untreated or treated with 2.4 mg/ml of mPPIg5 dendrimers for 3 hours before analysis for protease resistant PrP^Sc content by PK digestion and immunoblot. <b>B</b>) RML infected N2a cells were treated with increasing concentrations of mPPIg5 for 4 days before analysis by PK digest and immunoblot for PrP^Sc. Apparent molecular mass based on migration of protein standards is indicated for 17, 25, and 30 kDa in both panel A and B. n = 3.</p

Effect of NH₄Cl on FL PrP^Sc levels.

<p>22LN2a#58 cells were mock treated or treated with mPPIg5 (20 µg/ml), STI571 (10 µM) or suramin (200 µg/ml) in the presence or the absence of 2 mM NH₄Cl for 48 hours before analysis for FL PrP^Sc content via nSCA. The number of FL PrP^Sc containing cells in 20,000 mock treated control cells (- NH₄Cl) is taken as the 100% value on the Y axis. All subsequent results are calculated from this. Results marked a, b, c and d are all statically significantly different from one another (p<0.001); Analysed using a Two-way ANOVA for PrP^res, Dendrimer × NH₄Cl (F_4,88  = 7.99; P<0.001). Post-hoc analysis by Tukeys test; Error bars represent SD; n = 3.</p

Validation of nSCA.

<p><b>A</b>) N2a cells and <b>B</b>) ScN2a cells were inoculated for 1.5 hours with increasing dilutions of PrP^27–30. The infective media was removed and replaced with regular DMEM for an additional 3 hours before cells were repeatedly washed with PBS and 20,000 cells attached per well of an ELISPOT plate. Cells were examined via the nSCA and mSCA. Positive result is defined as >30 spots/20,000 cells. The software used to count spots (infected cells) cannot count accurately above 1000 spots so a dose response curve is not observed with the mSCA for the ScN2a cells inoculated with PrP^27–30. Error bars represent SD; n = 2.</p

mPPIg5 effect on subcellular distribution of PrP^C.

<p><b>A</b>) Confocal microscopy was used to examine the cellular localization of PrP^C. N2a#58 cells grown on coverslips were mock treated, treated with 20 µg/ml mPPIg5 or treated with 200 µg/ml suramin for 48 hours. Cells were then permeabilized or left intact before immuno-staining with SAF32 and Alexa488 secondary antibody for PrP^C. Hoechst 33342 was used to stain DNA. All images were acquired using equal settings. n = 2. <b>B</b>) Phosphatidylinositol-specific phospholipase C (PIPLC) was used to validate the confocal microscopy result. N2a#58 cells were treated with 20 µg/ml mPPIg5 (lanes 2, 4, 6 and 8) or mock treated (lanes 1, 3, 5 and 7) for 48 hours. Cells were washed with PBS then treated for two hours with 0.5U/ml PIPLC in serum free DMEM (lanes 3, 4, 7 and 8) or mock treated with serum free DMEM (lanes 1, 2, 5 and 6). The media was collected, ethanol precipitated and examined for PrP^C by immunoblotting with SAF83 antibody (lanes 5–8). The remaining cells were lysed and a portion examined for PrP^C by immunoblot (lanes 1–4). Apparent molecular mass based on migration of protein standards is indicated for 17, 25, and 30 kDa. <b>C</b>) Biosynthesis of PrP^C was examined by metabolic labelling. N2a cells were transfected with PrP^C and labelled with [³⁵S] methionine for 1 hour. After labelling cells were either analysed directly (Pulse), or incubated in fresh medium for additional 4 h (Chase). When indicated, mPPIg5 was present during the starving, labelling and chase periods (final concentration 20 µg/ml). PrP was immunoprecipitated by using the monoclonal anti-PrP antibody 3F4 and analysed by SDS-PAGE and autoradiography. Apparent molecular mass based on migration of protein standards is indicated for 16, 22, and 36 kDa.</p

Influence of Surface Groups on Poly(propylene imine) Dendrimers Antiprion Activity

Prion diseases are characterized by the accumulation of PrP^Sc, an aberrantly folded isoform of the host protein PrP^C. Specific forms of synthetic molecules known as dendrimers are able to eliminate protease-resistant PrP^Sc in both an intracellular and in vitro setting. The properties of a dendrimer which govern this ability are unknown. We addressed the issue by comparing the in vitro antiprion ability of numerous modified poly­(propylene-imine) dendrimers, which varied in size, structure, charge, and surface group composition. Several of the modified dendrimers, including an anionic glycodendrimer, reduced the level of protease resistant PrP^Sc in a prion strain-dependent manner. This led to the formulation of a new working model for dendrimer/prion interactions which proposes dendrimers eliminate PrP^Sc by destabilizing the protein and rendering it susceptible to proteolysis. This ability is not dependent on any particular charge of dendrimer, but does require a high density of reactive surface groups