U5 snRNP200 antibody–induced death is nonapoptotic. (A) Time-lapse phase contrast fluorescent imaging of THP-1 cells, labeled with calcein AM (green) and incubated with sAT1337 or the AML-binding, noncytotoxic IgG3 antibody sAT1412 in the presence of Cytotox Red to indicate dying cells (red). Stills were taken every 2 minutes. Dying cells lose calcein AM green and become Cytotox Red. Scale bars represent 25 μm. BF, bright field. (B) THP-1 cells incubated with sAT1223 led to increased membrane permeability (PI⁺) but not loss of mitochondrial membrane potential (DioC6⁺). Diclofenac, which induces apoptosis of THP-1 cells,¹⁴  was used as a positive control. Incubation with diclofenac led to loss of mitochondrial potential and increased membrane permeability (DioC6⁻ PI⁺). (C) Cell death of THP-1 cells by U5 snRNP200 complex–specific antibodies (incubated overnight) could not be blocked with the pan-caspase inhibitors Z-VAD-fmk or Q-VD-OPh. DMSO, dimethyl sulfoxide. (D) Stills of time-lapse imaging of 2 dying THP-1 cells, with frames taken every 2 minutes. Dying cells became adherent, flattened out, and lost membrane integrity, as indicated by loss of green calcein AM dye and uptake of Cytotox Red dye. Scale bars represent 25 μm. Graph indicates percentage of cells adhering to the plate at 15 and 60 minutes (n = 3). (E) Cell death and induction of typical morphologic changes by B cell–derived (sAT1337, sAT1331) and recombinant (rAT1337) antibodies (n = 3). (F) Preincubation of the target cells with the membrane-stabilizing agent cytocholasin D (in 0.1% DMSO) protected the THP-1 cells against cell death by AML-specific cytotoxic antibodies (left panel); however, this did not affect binding of AML-specific antibodies (right panel). Cells were incubated with antibodies for 4 hours.