Late Endosomal Cholesterol Accumulation Leads to Impaired Intra-Endosomal Trafficking
Figure 8
U18666A treatment protects cells against the action of ASSP.
AB: BHK cells were treated with or not with U18666A for 8 hrs, followed by 9 hrs treatment, still in the presence of the drug, with the mutant aerolysin toxin ASSP (500 ng/ml). B. For each conditions, the number of vacuolated cells, out of a total of n = 240, was counted. C: BHK cells were treated with trypsin nicked ASSP (500 ng/ml) either for 1 h at 4°C or for 24 h at 37°C. For cells treated at 4°C, 20 µg of total cell extracts were analyzed for the presence of monomeric and heptameric aerolysin by non-reducing SDS-PAGE and Western blotting. Cells treated at 37°C, were submitted to subcellular fractionation to isolate late endosomes, which were analyzed for the presence of monomeric and heptameric aerolysin by non-reducing SDS-PAGE and western blotting. Monomeric ASSP underwent differential migration when reduced in late endosomes vs. non-reduced at the cell surface as previously observed [22]. D: BHK cells were treated with U18666A for 8 hrs. Alexa-ASSP was subsequently added and allowed to be internalized for 6 hrs. Cells were fixed and processed for fluorescence microscopy to detect flotillin-1 in late endosomes.