Correlation of homotypic adhesion, actin reorganization, and lysosomal involvement to cell death evoked by the bispecific anti-CD20/CD74 HexAbs. (A) JeKo-1 cells were pretreated or not pretreated with the actin polymerization inhibitor, latrunculin B (1μM), for 2 hours followed by addition of HexAbs or both parental mAbs and evaluated for aggregation after 4 hours by light microscopy. (B) Apoptosis induced by HexAbs was reduced significantly (P < .025) in JeKo-1 with 1μM cytochalasin D (CsD), another inhibitor of actin polymerization. Error bars represent SD; n = 3. (C) Lysosomal V ATPase inhibitors, concanamycin A (Con A) and bafilomycin A1 (Bfa1), inhibited the apoptosis induced by HexAbs in JeKo-1 cells. (D) HexAbs induced changes in lysosomal volumes as determined by staining the cells with LysoTracker (75nM). The lysosomal compartmental volume changes were analyzed for JeKo-1 cells (top panel) and Granta-519 (bottom panel) by flow cytometry as enhanced binding and shift in the emission spectrum toward the right. (E) Immunofluorescence analysis of cathepsin B staining (red) 48 hours after treatment with select Abs. Nuclei were stained with DAPI (blue).