Immunoblotting HeLa or PC-3 cells (16106 cells) were incubated overnight and exposed to different concentrations of 3-azidoWA along with DMSO as vehicle. Cells were accordingly rinsed with PBS, trypsinized and collected after 24 h and then lysed with lysis buffer (HEPES 1.0 mM/L, KCl 60 mM/L, NP-40 0.3%, EDTA 1.0 mM/L, DTT 1.0 mM/L, Sodium orthovandate 1.0 mM/L, PMSF 0.1 mM/L, cocktail protease inhibitor). The cell extrac- tions were centrifuged at 12,000 rpm for 10 min at 4uC. Protein concentration was determined by the standard Bradford method. Equal amount (20 mg) of protein from each sample was subjected to SDS-PAGE and proteins were transferred to membrane (Millipore), blocked with 5.0% (w/v) non-fat milk in PBS containing 0.1% Tween-20 and probed with relevant antibodies for 3 h at room temperature or overnight at 4uC. Subsequently blots were washed and probed with species specific secondary antibodies coupled to horseradish peroxidise. Immunoreactive proteins were detected by enhanced chemiluminescence plus (Amersham). Apoptosis, Annexin V-FITC and PI Staining Following treatments with 3-azidoWA/vehicle DMSO, adher- ent cells (HeLa and PC-3) were harvested by trypsin digestion and washed twice with chilled PBS. Cells were fixed with 4% paraformaldehyde for 10 min followed by incubation with DAPI containing mounting medium for 15 min/RT in the dark and apoptosis was detected by fluorescence microscopy (100x magni- fication).The Annexin V-FITC experiments were performed using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer’s manual. Briefly, cell pellets were re-suspended in 600 ml binding buffer (10 mM HEPES [N-2- hydroxyethylpiper- azine-N-2–ethanesulfonic acid], 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4), and stained with 6.0 ml Annexin V-FITC and 10 ml propidium iodide staining solution for 20 minutes at room temperature in the dark. The samples were successively analyzed by FACS (BD FACS Aria II) using BD Diva software. Gelatin Zymography The gelatinase activity of MMP-2 was assessed by method standardized previously. Accordingly, HeLa and PC-3 cells in sub confluent culture (,70–80% cell density of confluent culture) were refreshed with new medium and kept for incubation with increasing concentrations of 3-azidoWA for 48 h. The conditional media obtained from both treated and untreated samples were employed for protein estimation and equal amount of total proteins (20 mg) were mixed with sample buffer (2.0% SDS, 25% glycerol, 0.1% Bromophenol blue and 60 mM Tris-HCl, pH 6.8). Gelatin zymography of the samples were carried out using 7.5% SDS-polyacrylamide gels containing 0.1% gelatine at 100V for 3 h at 4uC. Gels were accordingly rinsed with washing buffer (2.0% Triton X-100 in dd water) at room temperature to remove SDS followed by incubation overnight at 37uC in TCNB buffer (50 mM Tris-HCl, pH 8.0, 10 mM CaCl2, 0.02% NaN3). Gels were stained with Comassie blue R-250 (Sigma) (0.125% Comassie blue R-250, 50% methanol, 10% acetic acid) for 1– 1.5 h and destained with destaining solution (20% methanol, 10% acetic acid, 70% dd water). Gelatinase activity was detected by observing unstained bands on a blue background on Comassie stained gel. Matrigel Invasion Assay The effect of 3-azidoWA treatment on cell invasion was determined using BD Biocoat Tumor Invasion Assay System 3-azidoWA Blocks MMP-2 and Induces Secretory Par-4 (BD Bioscience, Bedford, MA) according to the instruction of the manufacturer. Briefly, HeLa and PC-3 (1.256106) cells were cultured in the presence of 0.25, 0.50, and 0.75 mM 3-azidoWA or vehicle DMSO for 24 h in serum free media into the upper chambers/inserts, and the bottom wells were filled with chemo- attractant (complete media with 10% FBS). Cells were allowed to migrate at 37uC. After 24 h the Matrigel-coated polycarbonate filters were removed, the non-migrating cells were separated from the upper chamber with a cotton swab and the insert were fixed with methanol and stained with 0.1% crystal violet solution. For each replicate (n = 3), migration of the cells was quantified by counting the stained cells (cells per five fields) under inverted microscope. Transient Transfection PC-3 and HeLa cells were cultured in appropriate medium as described in method section, transfected with GFP and GFP-Par-4 (generous gifts from Dr. Vivek Rangnekar, University of Kentucky, KY) using lipofectamine-2000, according to manufac- turer’s instruction. Forty eight hours post-transfection, green- fluorescence cells were visualized under fluorescence microscope with camera and conditioned medium was employed for gelatin zymography. Immunocytochemistry For immunostaining HeLa and PC-3 cells were plated on coverslips in 6-well plates at a seeding density of 0.5 x 106 cells per well. After 24 h, cells were treated with 3-azidoWA (1.0 mM) or vehicle DMSO and positive control staurosporine (25 nM) in presence or absence of Pan-Caspase inhibitor for 48 h. After the incubation, cells were washed with PBS and fixed in 2.5% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5.0 min and was successively blocked in 0.5% BSA for 1 h. For detection of Caspase activity, the cells were incubated with Caspase-3 primary antibody (1:5000 dilution in blocking buffer) for 1 h and accordingly washed three times with PBS, it was then incubated with Texas red (Invitrogen) conjugated secondary antibody (1:10000 dilution) for 1 h and washed, mounted with ultracruz mounting medium and analyzed with Zeiss LSM-510 metaconfocal microscope. The images were captured at 63x magnification. In vivo Matrigel Angiogenisis Assay Four to 6 week old C57/BL6J mice (Indian Institute of Integrative Medicine Central Animal House, Jammu, India) were maintained at 20–22uC on a 12 h light-dark cycle. Animal studies were performed in accordance with experimental protocols that were approved by the animal ethics committee of Indian Institute of Integrative Medicine, Jammu, India. Animals were injected subcutaneously, into the right flanks with 0.5 ml ice-cold Matrigel (BD Bioscience) supplemented with VEGF-A (250 ng/mL) and bFGF (500 ng/mL). Control mice were injected with Matrigel without VEGF-A and bFGF. At the end of each study animals were sacrificed to remove Matrigel plugs and photographs showing the extent of vascularisation was taken by using Nikon camera. The neovascularisation of Matrigel plugs was quantified by using 4 ml Drabkin’s reagent by adding well homogenated 20 ml of neovascularised Matrigel. After thorough mixing, absorbance was measured by spectrophotometer at wave length 540 nm to estimate hemoglobin. The hemoglobin estimation was calculated using formula Hb (g/dl) = absorbance of sample/ absorbance of standard 6 concentration of standard. A preliminary study was carried out to determine the time for optimal neovascularisation of Matrigel plugs to develop. In order PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44039