Inhibition of lysosomal exocytosis in injured cells results in defective endocytosis and plasma membrane repair. (A) β-Hexosaminidase (βHex) secretion from nontreated (NT) or BEL-treated NRK cells after SLO exposure with or without Ca²⁺. Error bars represent SD. (B) Nontreated or BEL-treated cells processed for EM 4 min after exposure to SLO/Ca²⁺ and BSA-gold. Short arrows point to BSA-gold particles within endosomes in nontreated cells (NT); long arrows point to swollen ER compartments in BEL-treated cells (BEL). M, mitochondria. (right) EM quantification. The data represent the mean ± SD (**, P < 0.0001; unpaired Student’s t test). (C) FACS quantification of WGA-FITC endocytosis after exposure to SLO/Ca²⁺ in nontreated or BEL-treated cells. (D) FACS quantification of PI staining in nontreated or BEL-treated cells permeabilized with SLO. Without Ca²⁺ (middle), both groups of cells remained permeabilized; with Ca²⁺, nontreated cells resealed their membrane more efficiently, excluding PI (right). (E) FACS quantification of PI staining in nontreated or BEL-treated cells after scrape wounding. Cells treated or not with BEL were similarly wounded, as indicated by the uptake of PI added during scraping (left). PI added 4 min after scraping revealed that both groups remained permeabilized without Ca²⁺ (middle), but with Ca²⁺, nontreated cells resealed their membrane more efficiently, excluding PI (right). Percentages correspond to resealed (PI negative) cells in the gated region (dashed lines). The results shown in this figure are representative of several independent experiments. Bars, 250 nm.