A coculture system reveals the functional significance of crosstalk between ECs and pericytes. (A,B) Quantitative RT-PCR analysis of Bcl-w (A) and VEGF-A (B) expression in MS1 endothelial cells cultured alone or together with HBVPs for 96 hours, either as 2D cultures allowing physical contact between ECs and pericytes or in transwell cultures where the 2 cell types are separated by a membrane with pore size 0.4 μm. Data shown represent the mean of 9 and 2 independent experiments (2D cultures and transwell cultures, respectively). *P < .05 vs MS1 mono-culture. (C) Western blot (WB) analysis of Bcl-w and VEGF-A in cell lysates from MS1 endothelial cells cultured alone or together with HBVPs for 96 hours. M_r indicates molecular weight (kDa). Western blot for β-actin was used as a loading control. Dotted line marks the removal of one irrelevant lane from the original photograph. (D) Quantitative RT-PCR analysis of Bcl-w expression in MS1 endothelial cells cultured alone or together with HBVPs for 96 hours with the addition of the VEGF inhibitors sFlt1 (a ligand trap acting extracellularly) or AG-028262 (an intracellular inhibitor blocking VEGF signaling via its receptor tyrosine kinases). *P < .05 vs MS1 mono-culture; **P < .01 vs MS1/HBVP coculture. (E) Quantification of the number of viable MS1 endothelial cells cultured alone or together with HBVPs for 96 hours with the addition of the cytotoxic drug vinblastine (50 ng/mL) and the VEGF inhibitors sFlt1 (blocking only extracellular sources of VEGF-A) or AG-028262 (blocking both extra- and intracellular sources of VEGF-A). *P < .05 vs vinblastine-treated coculture; **P < .01 vs vinblastine-treated mono-culture; and §, not statistically significant vs vinblastine-treated coculture. HBVP indicates human brain vascular pericytes.