Plasma cell apoptosis is largely caspase-independent. (A) Apoptosis of ex vivo–isolated and in vitro–differentiated plasma cells and I.29μ⁺ cells. The proportion of dead cells was determined by staining with annexinV-FITC and propidium iodide. Primary and in vitro–differentiated plasma cells were analyzed 1 day and 2 days after isolation, respectively. I.29μ⁺ cells were analyzed before (d0) and one to 4 days after stimulation of plasmacytic differentiation with LPS. Mean and SEM of 2 independent experiments are shown. Statistically significant differences between means are indicated by an asterisk. (B) Immunoblotting on whole cell extracts prepared at the same time points as described in (A). Extracts from primary splenic resting B cells (RB) were prepared after one day in culture following ex vivo isolation. (C) Primary ex vivo–isolated and in vitro–differentiated plasma cells were treated with zVADfmk (100μM) or DMSO immediately after isolation for 24 and 48 hours, respectively. I.29μ⁺ cells were treated from day 2 to 4 after LPS stimulation. The proportion of apoptotic cells was determined as in panel A. (D) Immunoblotting with antibodies against the indicated proteins after treatment with tunicamycin (1 μg/mL) for 14 hours. Images are from the same blot. For comparison with untreated (control) cells see panel B. (E) Ex vivo–isolated resting B and plasma cells, undifferentiated I.29μ⁺ cells, and in vitro–differentiated plasma cells were treated with tunicamycin (1 μg/mL) and zVADfmk (100μM) or DMSO (control) for 14 hours, and the proportion of apoptotic cells determined as in panel A. Note that tunicamycin was used at 5 μg/mL in resting B cells, and treatment was extended to 24 hours in I.29μ⁺ cells to achieve comparable rates of apoptosis. (F) Analysis of mitochondrial outer membrane permeabilization during plasma cell apoptosis by immunoblotting of cytosolic and whole cell extracts from undifferentiated (d0) and plasmacytic (d3) I.29μ⁺ cells with antibodies against cytochome C and GAPDH (control).