Figure 4

Prevention of secretion of either cell component of coculture using BFA. (A) BFA treatment (8 h) reduced VEGF protein secretion from 3905.4 ± 251.4 pg/mL (untreated control (UTC)) to 68.4 ± 21.3 pg/mL and from 193.4 ± 12.6 pg/mL (UTC) to 34.8 ± 10.9 pg/mL in stromal cells (left) and CLL cells (right), respectively (P = 0.002 and P = 0.01, DMSO versus BFA treatment for stromal cells and CLL cells, respectively, paired two-tailed t test). Secreted VEGF was analyzed by ELISA. (B) Survival was 83.4% ± 2.4% (UTC) and 76.3% ± 1.6% (BFA) for stromal cells (left) and 73.0% ± 2.9% (UTC) and 69.3% ± 3.1% (BFA) for CLL cells (right). (C) Coculture with BFA-treated stromal cells provided a significantly reduced survival advantage (2.3% ± 3.0%) when compared with coculture with untreated stromal cells (11.3% ± 5.7%) (P = 0.038, paired two-tailed t test). (D) BFA-treated CLL cells on untreated stromal cells did not show significantly reduced survival when compared with an untreated CLL cell survival in a coculture setting (4.1% ± 1.4% versus 5.5% ± 1.1%). DMSO functioned as a vehicle control. Survival advantage was calculated by subtracting survival (%) in monoculture from survival (%) in coculture. Survival was assessed using annexin V-FITC/PI staining on FACSCanto. *P < 0.05; **P < 0.01; n.s., not significant.