Induction of APRIL accumulation in the ER of B-CLL cells following brefeldin A treatment. (A) APRIL accumulates in the ER following treatment with brefeldin A. Primary CLL cells were treated with 100 ng/mL BFA for 24 hours. Following drug treatment, cells were cytocentrifuged onto glass slides and fixed as described in “Patients, materials, and methods.” Cells were stained with a monoclonal anti-APRIL antibody and markers for the ER (calreticulin) and nucleus (To-Pro-3). APRIL immunofluorescence was visualized by confocal microscopy. Images were obtained as for Figure 3A. (B) Quantitation of the BFA-induced increase in APRIL immunofluorescence. The intensity of fluorescence was quantitated using Optimas software as described in “Materials and methods.” n = 8 for each condition; error bars indicate the SD. (C) Brefeldin A causes an increase in the intracellular levels of the APRIL protein. Primary CLL cells were treated with 100 ng/mL BFA for 24 hours. Lysates from control and drug-treated cells were electrophoresed, and APRIL levels were assayed by immunoblotting with an APRIL-specific antibody. Actin was used as a loading control. NR = nonrefractory; R = refractory.