Production of ROS occurs downstream of mAb-induced HA, actin-reorganization and lysosome permeabilization. (A) Raji cells were treated with PBS or tiron (10mM) for 2 hours before addition of mAb (10 μg/mL). Four hours later cells were assessed for HA by light microscopy. Original magnification ×20. (B) Cells were treated as in panel A, cytospun on poly-L-lysine coated slides and stained with Alexa Fluor 488–Phalloidin to label the actin filaments and slides were assessed by fluorescence microscopy. Representative images are shown. Scale bar: 25 μm. Tiron does not affect mAb-induced relocalization of actin filaments to the cell-cell junctions. (C) Raji cells were treated with the actin inhibitor latrunculin B (Lat B; 10μM) for 30 minutes before the addition of mAb, as before. Samples were assessed for ROS production after HE staining and flow cytometry. Bars represent mean + SEM from 3 separate experiments. (D) Cells were labeled with AO, and then treated with PBS or tiron and mAbs as before. The relative increase in green fluorescence, indicative of leakage of AO from the acidic lysosomes into the more pH neutral cytosol was assessed by flow cytometry 4 hours after addition of mAbs. Bars represent mean + SEM from 3 separate experiments. (E) Immunofluorescence staining of the lysosomal protease cathepsin B after mAb treatment in the presence or absence of tiron. Cells were treated and stained as described in panels A and B. Representative images are shown. Scale bar: 50 μm. Tiron does not block mAb-induced cathepsin B release into the cytoplasm and areas of cell-cell contact. (F) Cells were treated with cathepsin inhibitor III (100μM) 30 minutes before addition of mAbs (10 μg/mL). Samples were assessed for ROS production 4 hours later after HE staining and flow cytometry. Bars represent mean + SEM from 3 separate experiments. Together these data indicate that production of ROS occurs downstream of mAb-induced HA, actin reorganization and lysosomal membrane permeabilization.