VEGF trapping up-regulates MMP-2 expression in glioma cell lines. (A) Western blot of U87, U118, A172 and U251 glioma cells after treatment with VEGF-Ab for 5 days. Before collection, cell secretion was blocked for 6 hours with Golgi-Plug. Human β-Actin was used as a protein loading control. (B) Quantitative real-time PCR for MMP-2 mRNA levels in glioma cell lines was evaluated using the ΔCT method. Before plotted in the graph, the MMP-2 expression for each cell line was normalized to their Ig-Control treated condition, to which was given an arbitrary value of 1 (dotted, thin black line: Control). The relative expression levels of MMP-2 for all four cell lines upon VEGF-Ab treatment were then presented as fold of their Control treated cells at the indicated time points. (C) Immunocytochemistry (ICC) of glioma cell lines for MMP-2 and collagen IV after treatment with VEGF-Ab. Cells treated in vitro with VEGF-Ab for 5 days were blocked with Golgi-Plug for 6 hours before fixation for ICC. Nuclei are stained blue with DAPI; collagen IV is presented in yellow and MMP-2 in red. Bars equal 50μm.