PFN and GzmB are rapidly endocytosed into EEA-1⁺ outsized early endosomes. (A-B) Within 5 minutes of treatment with sublytic native hPFN and hGzmB, large intracellular and membrane bound endosomes (gigantosomes) coimmunostain for GzmB (A) or PFN (B) and the early endosomal marker EEA-1. Images were acquired by wide-field fluorescence microscopy and deconvolved using iterative deconvolution. Representative Z stack series projections from 3 independent experiments are shown. Percentage of cells with GzmB or PFN staining enlarged endosomes is indicated (mean ± SD). (C) HeLa cells were exposed to sublytic hPFN for indicated times and stained for PFN and EEA-1. PFN is first detected at the plasma membrane and then endocytosed into EEA-1⁺ endosomes before forming gigantosomes. Representative confocal sections from 3 independent experiments are shown. PFN or GzmB was detected using AlexaFluor 488–conjugated secondary antibody and EEA-1 using AlexaFluor 647–conjugated secondary antibody. Color bars and associated numbers indicate fluorescence intensity levels. Scale bars represent 10 μm. Dashed lines indicate plasma membrane. Magnification and other image acquisition data are in supplemental Methods.