Perforin increases clathrin-mediated endocytosis. (A) Within 7 minutes of treatment, sublytic rPFN and SLO activate uptake of A488-GzmB, whereas ionomycin, even at the highest lytic concentration, does not. Mean fluorescence intensity (mean ± SD) from 3 independent experiments is indicated. (B) rPFN and SLO increase the rate of AP-2–dependent endocytosis, but ionomycin does not. HeLa cells stably expressing EGFP–AP-2σ2 were used for spatial and temporal analysis of AP-2 at CCPs. Cells were imaged every 10 seconds by spinning disk confocal microscopy before and after addition of sublytic rPFN, ionomycin, or SLO. The maximum fluorescence intensity and lifetime of new membrane-associated AP-2 spots were measured 400 seconds before and after treatment. Representative data from one cell treated with rPFN, ionomycin, or SLO (supplemental Videos 3-5) are shown. (C-D) Sublytic rPFN and SLO significantly increase the number of new AP-2 spots associated with the plasma membrane and the total intensity of membrane-associated AP-2 molecules, whereas ionomycin (5μM) only slightly increases membrane-associated AP-2. Data depicted were obtained from 3 different cells and 3 independent experiments. Data depicted in panel B correspond to cell no. 3 for each treatment. (E) The percentage increase of AP-2–mediated endocytosis after treatment (mean ± SD) was calculated. (F) Intracellular Ca²⁺ increases after sublytic rPFN, SLO, and ionomycin treatment. Calcium influx was measured by FlexStation III (Molecular Devices) in HeLa cells stained with Fura-2/AM at 5-second intervals after adding PFN, SLO, or ionomycin. At sublytic concentrations, rPFN and SLO induce a transient Ca²⁺ flux, whereas ionomycin induces a sustained and global rise of intracellular Ca²⁺. Data are representative of 3 independent experiments.