JCS02609 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Schematic representation of MTT assay upon treatment of 3F4-ScN2a with suramin derivatives for 4 days. Cell viability of 3F4-ScN2a was measured on 96-well microtitre plates upon incubation with suramin derivatives for 4 days at a concentration of 200 mg/ml. Optical density value measured for mock-treated cells was taken as 100% viability. Each bar represents the mean cell viability (including s.d.) of compound-treated cells as a percentage of the values collected for mock-treated cells. Measurements were recorded in triplicate.

• Supplemental Figure 2 -

  Fig. S2. Low doses of suramin and derivatives are less effective in reducing the amount of PrP^Sc in prion-infected cells. 3F4-ScN2a cells were seeded on 60 mm plates and treated daily with suramin or with selected derivatives. All compounds were applied at a concentration of 50 or 100 mg/ml for 4 days. Mock-treated cells were used as a control. Cells were harvested and postnuclear lysates were either treated with 20 mg/ml PK for 30 minutes at 37°C or subjected to ultracentrifugation at 100,000 g in the presence of 1% sarcosyl, to separate soluble (S, supernatant) from insoluble (P, pellet) fractions. PrP was visualised by immunoblotting using the monoclonal anti PrP antibody 4H11. Molecular size markers are indicated on the left. PK-resistant PrP can still be detected after treatment of the cells with some of the suramin derivatives in a dose- and time-dependent manner. Actin loading controls are shown underneath each panel.

• Supplemental Figure 3 -

  Fig. S3. Three-day treatment of prion-infected cells with suramin or its derivatives is less effective in reduction of PrP^Sc to undetectable levels. 3F4-ScN2a cells were treated daily with suramin or with selected derivatives. All compounds were applied at a concentration of 200 mg/ml for 3 days. Cells were processed as described in supplementary Fig. S2. Molecular size markers are depicted on the left. PK-resistant PrP can still be detected after treatment of the cells with some of the suramin derivatives.

• Supplemental Figure 4 -

  Fig. S4. Chimeric PrP-Lamp is not expressed at the cell surface and localises in lysosomes. (A) Schematic representation of the chimeric protein PrP-Lamp: the GPI-attachment signal of the murine PrP (residues 232-254) was substituted by the transmembrane and cytoplasmic segment of the lysosomal protein LAMP-1 (residues 348-382). The construct carries the 3F4 epitope. (B) The intracellular and surface expression of PrP-LAMP were measured by FACS analysis upon transient transfection into N2a cells. Upper panel shows fluorescence histograms for intracellular expression of PrP-LAMP in permeabilised cells for one representative experiment. x-axes represent the fluorescence intensity plotted against the number of cells (y-axes). In each experiment, the expression in transfected cells (dotted line) was measured against the one in nontransfected ones (solid line). For each population 10,000 cells were counted. Lower panels show cell surface expression in non-permeabilised cells. Living and dead cells were separated by staining with propidium iodide and gating. In both assays the monoclonal antibody 3F4 was used for detection of the transfected construct. (C) Confluent N2a cells transiently transfected with wtPrP or PrP-LAMP were treated for 48 hours with 200 mg/ml suramin (untreated cells were used as a control) and were then biotinylated for 15 minutes on ice to examine surface expression of the transfected proteins. Cells were then lysed and wtPrP and PrP-LAMP were precipitated with monoclonal antibody 3F4. Samples were subjected to SDS-PAGE analysed by immunoblotting. Biotinylated proteins were detected with streptavidin. A specific signal was detected for biotinylated wtPrP (lane 2), which disappeared upon treatment with suramin (lane 1). PrP-LAMP could not be biotinylated (lanes 3 and 4), confirming that the protein is not localised at the cell surface.

• Supplemental Figure 5 -

  Fig. S5. Chimeric PrP-Lamp localises in lysosomes and Golgi compartment. N2a cells were transiently transfected with wt3F4 PrP or PrP-Lamp and the intracellular localisation of these proteins was analysed 72 hours post transfection in a confocal microscopy study. Staining of 3F4PrP (panels a-f) and PrP-Lamp (panels g-l) was performed using the monoclonal antibody 3F4. Lysosomes and the Golgi compartment were visualised with the acidotropic amine DAMP (panels e,k) or rabbit anti-Rab 6 antibody (panels e,h), respectively. PrP-Lamp was mainly detected intracellularly, in endosomal/lysosomal compartments (panels j-l) and the Golgi (panels g-i), whereas wtPrP also localised on the cell surface (panels a,d). Right-hand panels show an overlay of both signals.