The defects in endocytosis and plasma membrane repair observed after silencing of ASM expression are rescued by exogenous rhASM. (A) ASM activity in lysates of HeLa cells treated with control or ASM siRNA. Error bars represent SEM. SM, sphingomyelin. (B) Immunoblots with anti-ASM (top) or antiactin antibodies (bottom) of HeLa cells not treated or treated with control or ASM siRNA. The arrow points to the ∼75-kD band corresponding to ASM. The higher additional band is an unspecific reaction. (C) Quantification of BSA-gold–containing endosomes detected by EM in HeLa cells 4 min after exposure to SLO/Ca²⁺ and BSA-gold. The cells were pretreated with control siRNA, ASM siRNA, or ASM siRNA followed by rhASM added during the SLO wounding procedure. The data represent the mean ± SD (**, P < 0.0001; comparing control or ASM + rhASM with ASM by unpaired Student’s t test). (D) Time-lapse imaging of FM1-43 influx into HeLa cells exposed or not to SLO in the presence of Ca²⁺. FM1-43 influx was contained in cells treated with control siRNA but not in cells treated with ASM siRNA. The addition of rhASM at the time of SLO permeabilization restored the capacity of NPA cells to stop FM1-43 influx (Video 2). Non–SLO-permeabilized cells did not show a significant increase in FM1-43 intracellular staining (green and purple). 37–63 cells were analyzed in each condition; error bars correspond to the mean ± SEM. (E) Selected time frames of Video 4. Bars, 9 µm.