Figure 4.

Vacuolin-1 does not inhibit the surface exposure of Lamp-1 induced by plasma membrane wounding. (A) Confluent monolayers of HeLa cells grown on glass coverslips were treated or not with 5 μM vacuolin for 1 h, scratched several times in a grid fashion with a scalpel, transferred to ice and surface labelled with anti-Lamp-1 monoclonal antibodies. After fixation, cells were incubated with a fluorescent secondary antibody (green), and 4,6-diamidino-2-phenylindole to stain cell nuclei (blue). The arrows point to wounded cells that exposed the luminal domain of Lamp-1 on the cell surface. (B) NRK cells, treated or not with vacuolin-1 for 1 h, were wounded in suspension by electroporation, surface stained for Lamp-1 and examined by fluorescence-activated cell sorting. No inhibition of injury-induced lysosomal exocytosis was observed after vacuolin-1 treatment. FL1-H, fluorescence intensity; FSC-H, forward scatter; SSC-H, side scatter.