Blocking ASM activity does not inhibit lysosomal exocytosis but impairs endocytosis and plasma membrane repair. (A) ASM activity in NRK cell lysates not treated (NT) or treated with DPA (95% inhibition). Error bars represent SEM. SM, sphingomyelin. (B) β-Hexosaminidase (βHex) secretion from nontreated (NT) or DPA-treated NRK cells after exposure to SLO with or without Ca²⁺. Error bars represent SD. (C) Quantification of BSA-gold–containing endosomes detected by EM in DPA-treated or nontreated cells 4 min after exposure to SLO/Ca²⁺ and BSA-gold. The data represent the mean ± SD (**, P < 0.0001; unpaired Student’s t test). (D) FACS quantification of WGA-FITC endocytosis in nontreated or DPA-treated NRK cells after SLO exposure or scraping in Ca². (E) Time-lapse imaging of FM1-43 influx into NRK cells exposed or not to SLO in the presence of Ca²⁺. FM1-43 influx was contained in nontreated cells (black) but not in DPA-treated cells (red; Video 1). Cells not exposed to SLO did not show a significant increase in FM1-43 intracellular staining (green and purple). 30–89 cells were analyzed in each condition; error bars correspond to the mean ± SEM. (F) Selected time frames of cells treated or not with DPA but not exposed to SLO and of Video 1 (SLO-exposed cells treated or not with DPA in Ca²⁺). The results shown in this figure are representative of several independent experiments. Bars, 9 µm.