ASM-deficient NPA fibroblasts are defective in injury-dependent endocytosis and plasma membrane repair. (A) ASM activity in lysates of normal human fibroblasts (NC) or NPA human fibroblasts. Error bars represent SEM. The inset shows an immunoblot of normal human or NPA fibroblasts using anti-ASM antibodies. The ∼75-kD band corresponding to ASM (arrow) was undetectable in NPA cells; the higher additional band is an unspecific reaction. SM, sphingomyelin. (B) β-Hexosaminidase (βHex) secretion from normal human or NPA cells after exposure to SLO with or without Ca²⁺. Error bars represent SD. (C) FACS quantification of endocytosis after scrape wounding in normal human or NPA cells. (D) Time-lapse imaging of FM1-43 influx into cells exposed or not to SLO in the presence of Ca²⁺. FM1-43 influx was contained in normal human cells (Video 2) but not in NPA cells. The addition of rhASM at the time of SLO permeabilization restores the capacity of NPA cells to stop FM1-43 influx (Video 3). Non–SLO-permeabilized cells did not show a significant increase in FM1-43 intracellular staining (purple). 4–11 cells were analyzed in each condition; error bars correspond to the mean ± SEM. (E) Selected time frames of Videos 2 and 3. The results shown in this figure are representative of several independent experiments. Bars, 9 µm.