Clathrin-mediated endocytosis is required for PFN-mediated GzmB internalization. (A-B) Inhibition of Dyn/clathrin-mediated endocytosis by pretreatment with hypertonic sucrose (300mM) or Dynasore (80μM) decreases sublytic rPFN-induced A488-GzmB internalization. (C) Cells transfected with Dyn2, AP-2μ2, or CHC siRNAs show reduced GzmB internalization, compared with control cells treated with GFP (ctrl) or Flotillin-1 siRNAs, 5 minutes after incubation with sublytic rPFN and A488-GzmB. Graphs are representative of 3 independent experiments and mean fluorescence intensity (MFI; mean ± SD) is indicated. (D) Concentration of A488-GzmB (green) in the nucleus of target cells 20 minutes after sublytic rPFN, and A488-GzmB incubation is seen in cells treated with flotillin-1 or GFP (Ctrl) siRNAs but not in cells treated with CHC siRNA. Numbers indicate the percentage of cells with nuclear GzmB (mean ± SD from 3 independent experiments). GzmB signal 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. Dashed lines indicate nuclei. Scale bar represents 10 μm.