Inhibiting clathrin-mediated endocytosis increases PFN-induced necrosis. Inhibition of Dyn/clathrin-dependent endocytosis increases necrotic cell death after sublytic rPFN treatment. HeLa cells were transfected with siRNAs for Dyn2, AP-2μ2, and CHC or pretreated with Dynasore (80μM), an inhibitor of Dyn GTPase function, and then incubated for 15 minutes with sublytic rPFN. GFP (ctrl) or Flotillin-1 siRNAs were used as negative controls. Necrosis was evaluated immediately by flow cytometric measurement of PI uptake. Representative histograms after sublytic rPFN treatment (A), and mean ± SD from 3 independent experiments with sublytic (B) or different doses of rPFN (C) are shown. *P < .03, #P < .001, relative to control siRNA–treated cells.