3-azidoWA Blocks MMP-2 and Induces Secretory Par-4 Figure 4. 3-azidoWA induced extracellular Par-4 abrogates invasion of PC-3 cells. (A) PC-3 Cells were left untreated or pretreated with BFA (1.0 mM) for 120 min, then treated with 3-azidoWA as indicated for 48 h. Conditioned media were prepared, and subjected to gelatin zymography for MMP-2 activity and western blot (WB) analysis for Par-4, followed by Coomassie blue staining for loading control. (B) PC-3 cells were left untreated or pretreated with BFA (1.0 mM) as indicated, then exposed to TRAIL (as indicated). Conditioned media were prepared, and subjected to gelatin zymography for MMP-2 and western blot (WB) analysis for Par-4. (C) & (D) Cell migration was determined by the modified Boyden chamber assay as described in materials and methods. HeLa cells (26105 cells/well) were seeded in top chamber in the presence of 0.25 and 0.50 mM of 3-azidoWA along with untreated in upper and lower lane. Cells were allowed to migrate for 24 h, at which point migratory cells on the bottom half of the insert membrane were stained with 0.1% crystal violet and counted under 200x magnification and in lower lanes Par-4 antibody was added in 0.25 and 0.50 mM 3-azidoWA treatment group and control PTEN antibody was added in untreated group and further incubated for 24 h. Migratory cells were counted using image software as the number of migrated cells per high-power field (HPF). Five fields were counted in triplicate from each insert of three independent experiments (SD. *, P,0.05). Migrated cell images were obtained using microscope Nikon Eclipse E200 inbuilt camera (200x magnification). (E) Zymography was performed from conditional media collected from cells treated with 3-azidoWA (0.25 and 0.50 mM) along with untreated and 3-azidoWA (0.25 and 0.50 mM) + Par-4 antibody, corresponding untreated + PTEN antibody. Coomassie stain of BSA confirms equal loading (lower band). (F) PC-3 cells were transiently transfected with GFP and GFP-Par4, 48 h post-transfection conditioned media were analyzed through gelatin zymography, followed by coomassie blue staining to reveal the 68 KDa BSA band for loading control and green fluorescence protein expressions were checked under fluorescence microscope and photographed (100x). Columns mean; bars SD of three independent experiments. *P,0.05, compared with untreated control. doi:10.1371/journal.pone.0044039.g004 3-azidoWA Inhibits in vivo Angiogenesis Tumor angiogenesis provides oxygen, nutrients, and main routes for tumor growth, invasiveness, and metastasis and acts as a rate-limiting step in tumor progression [35]. Initial study was carried out to determine the time required for optimum neovascularisation of Matrigel lacking VEGF and bFGF. As shown in (Fig. S3, A) for animals injected with Matrigel with out VEGF and bFGF, minimal vascularisation was noticed and there PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e44039