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Fig. 4. Treatment with suramin analogues does not downregulate cell surface expression of PrPc. (A) Expression levels of PrPc in 3F4-N2a cells treated for 3 days with 200 µg/ml NF023, NF449, NF023 or ANTS was measured by FACS analysis. The histograms depict the cell surface expression in non-permeabilised cells (upper panels) and the intracellular PrP expression in cells permeabilised with saponin buffer. FL1 represents the fluorescence intensity in treated cells (bold line) and in the mock-treated ones (dotted line), plotted against the number of cells (counts). For each cell population 10,000 events were measured. Living and dead cells were separated by staining with propidium iodide and gating. The monoclonal antibody 3F4 was used for detection of PrPc. No significant change in the intracellular or in the surface expression of PrPc was detected upon treatment of cells with any of the tested compounds. (B) PrPc localisation upon exposure to NF023 or NF449 was confirmed by confocal microscopy studies and compared to mock-treated cells in wtN2a cells, stably overexpressing PrPc. Tested compounds were added daily (48 hour treatment) to the culture medium at a concentration of 200 µg/ml, before fixation and permeabilisation. Staining of PrP and lysosomes was performed using the polyclonal anti-PrP antibody A7 (left-hand column) or a monoclonal antibody directed against the lysosomal protein LAMP-I (middle column), respectively. After treatment with NF023 or NF449, PrPc could still be detected on the cell surface (middle and lower left panels) at levels comparable to untreated cells (mock). Upon treatment with suramin derivatives, PrPc also shows increased localisation in intracellular compartments which partially correspond to lysosomes, as seen in an overlay of the two signals (merge). Bar, 10 µm.
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