Brefeldin A inhibits VEGF secretion. (A) Brefeldin A treatment caused VEGF to accumulate in the ER of primary CLL cells. CLL cells were treated with 100 ng/mL BFA for 24 hours. Cells were cytocentrifuged onto glass slides and fixed as described in “Materials and methods.” Fixed cells were stained with an antibody against VEGF and markers for the ER (calreticulin) and nucleus (To-Pro-3). The fluorescence of stained cells was visualized by confocal microscopy. Images were obtained as for Figure 3A. (B) Quantitation of the brefeldin A-induced increase in VEGF immunofluorescence. The intensity of immunofluorescence of multiple cells was quantitated for the control and BFA-treated cells using Optimas software as described in “Patients, materials, and methods” (n = 8). Error bars indicate the SD. RFU indicates relative fluorescence units. (C) Brefeldin A causes a dose-dependent reduction in VEGF secretion. Primary CLL cells were treated with 30 and 100 ng/mL BFA for 16 hours. Secreted levels of VEGF were quantitated in the culture medium by ELISA as described in “Patients, materials, and methods.” Bars represent the average of 2 assays, which varied by less than 10%.