Anti-CD20/CD74 HexAbs induce stress- and caspase-independent apoptosis in target cells. (A) Western blot analysis of select antiapoptotic and proapoptotic proteins on treatment with 10nM of indicated Abs for 48 hours in JeKo-1, Granta-519, and Mino cells. The 2 anti CD20/CD74 HexAbs down-regulated the antiapoptotic protein Bcl-xL. (B) Neither 20-(74)-(74) nor 74-(20)-(20) activated caspase 3, caspase 8, and caspase 9 in JeKo-1. (C) Caspase inhibitor z-vad-fmk did not inhibit the apoptosis in JeKo-1 induced by anti-CD20/CD74 HexAbs. JeKo-1 cells were pretreated with z-vad-fmk pan caspase inhibitor (10μM) for 2 hours followed by treatment with 10nM of indicated Abs or anti-IgM (10 μg/ mL, a positive control for caspase-dependent apoptosis) for 72 hours, and apoptosis was determined by annexin V staining. Error bars represent SD; n = 3. (D) Both anti-CD20/CD74 HexAbs induced changes in mitochondrial membrane potential (top panel) and generated ROS (bottom panel) in Granta-519 cells.