Figure 6

MMP-9 and Zo-1 expression and distribution in hCMEC/D3 cells treated with TWEAK. (A) Colabeling immunocytochemistry showing expression and distribution of MMP-9 and ZO-1 in control (CTRL), TWEAK-, and TNFα-treated hCMEC/D3 cells. MMP-9 showed a punctuate, vesicular-like pattern distributed throughout the cytoplasm that was increased in the TWEAK- and TNFα-treated cells. Superimposition of MMP-9 and ZO-1 images shows colocalization of both proteins (Merge (×20), scale bar 10 μm) in yellow-orange along the plasma membrane, visible as distinct puncta (arrows in Merge (×40), scale bar 2.5 μm). Note the global down-regulation of ZO-1 expression in TWEAK and TNF-treated hCMEC/D3 cells as compared with CTRL. (B) Confocal analysis of a single z-axis plan of the TWEAK-treated cells shows nuclear accumulation of MMP-9. Scale bar 2 μm. (C) Kymographs were constructed and analyzed with ImageJ software from single-pixel width lines taken from each channel of the confocal images in A (Merge (×40)). Profiles of the signal intensities of MMP-9 (green line) and ZO-1 (red line) measured along the single-pixel width lines drawn in Merge (×40) indicate colocalization. (D) Western blot analysis of protein extracts from TWEAK-treated hCMEC/D3 cells following separation by SDS PAGE and transfer onto nitrocellulose membranes, which were probed with primary antibodies against ZO-1 and actin. Both TWEAK and TNF treatments diminish ZO-1 expression in the hCMEC/D3 cells. Representative Western blot of three independent experiments.