On Jagged-1 protein expression in Hep G2 cells. Hep G2 cells were seeded at a density of 80,000 cells/ml in 6-well plate at day 0 in DMEM media supplemented with 10 FBS and penicillin/streptomycin mixture. Following day after seeding, the cells were treated with modified SL2-B aptamer and scrambledTable 1. Unmodified and ITI214 web PS-modified SL2-B aptamer sequences along with their equilibrium dissociation constant (Kd) values determined using surface plasmon resonance (SPR) spectroscopy.Sequences of original and PS-modified aptamer (59?9) Unmodified SL2-B aptamer CAATTGGGCCCGTCCGTATGGTGGGT PS-modified SL2-B aptamer C*AATTGGGCCCGTCCGTATGGTGGG*T “*” indicates the position of phosphorothioate (PS) modification. doi:10.1371/journal.pone.0050964.tKd 0.5060.32 nM 0.5660.44 nMAntiproliferative Activity of Aptamer on CancerFigure 2. SPR sensorgrams demonstrating interaction of PS-modified SL2-B aptamer with immobilized VEGF165 and VEGF121 protein at same concentration. Point A to B corresponds to association phase and point B to C corresponds to the dissociation phase in both the sensorgrams. Shown here is PS-modified SL2-B aptamer IT1t web binding with VEGF165 protein (Kd = 0.5660.44 nM) and VEGF121 protein (Kd = 1761.24 mM) at 80 nM aptamer concentration. doi:10.1371/journal.pone.0050964.gaptamer sequence at 15 mM aptamer concentration. Same hypoxia conditions were maintained as for the antiproliferative activity assay. After 3 days of aptamer treatment, the cells were trypsinized, incubated with anti-human Jagged-1 fluorescein antibody for 1 hour, re-suspended in PBS buffer and analyzed immediately using a Beckman-Couter CyAn ADP flow cytometer by analyzing 15,000 events and relative fluorescence was determined using SUMMIT V 4.3.02 software.bands were developed with west pico chemiluminescence substrate and visualized on XPress CL blue ray film. Optical densities of bands were measured on 1531364 a GS800 densitometer and band intensities were analyzed with Quantity One image analysis software (Biorad, USA).Statistical AnalysisData are presented as mean 6 SD. A p-value ,0.05 was considered statistically significant using student’s t-test.Western Blot AnalysisThe sequence specific effect of PS-modified SL2-B aptamer on Jagged-1 protein expression in Hep G2 cells was analyzed using western blotting. Same experimental conditions were maintained as for the flow cytometry. After 3 days of aptamer treatment, the cell medium was removed and the cells were washed once in cold 16PBS. 500 ml of the complete lysis buffer was added to each 6well and the cells were scrapped with a cell scrapper and collected into microcentrifuge tubes. The extracted proteins were resolved on an SDS-PAGE gel and transferred onto a PVDF membrane via wet transfer. Membranes were blocked in 5 non-fat milk and washed in tris-buffered saline with 1 tween. Subsequently, membranes were incubated with primary antibody (Jagged-1 rabbit monoclonal and purified mouse anti-calnexin antibody) and then with corresponding secondary antibody (goat anti-rabbit and anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (HRP)) with 3 washing steps in between. The proteinResults and Discussion Binding Analysis of PS-modified SL2-B Aptamer and VEGF Complex by Surface Plasmon Resonance (SPR)As reported previously in our study, the unmodified SL2-B aptamer displayed a Kd = 0.5 nM to heparin binding domain (HBD) of VEGF165 protein determined via SPR technique (Table 1) [37]. The unmodified aptamer,.On Jagged-1 protein expression in Hep G2 cells. Hep G2 cells were seeded at a density of 80,000 cells/ml in 6-well plate at day 0 in DMEM media supplemented with 10 FBS and penicillin/streptomycin mixture. Following day after seeding, the cells were treated with modified SL2-B aptamer and scrambledTable 1. Unmodified and PS-modified SL2-B aptamer sequences along with their equilibrium dissociation constant (Kd) values determined using surface plasmon resonance (SPR) spectroscopy.Sequences of original and PS-modified aptamer (59?9) Unmodified SL2-B aptamer CAATTGGGCCCGTCCGTATGGTGGGT PS-modified SL2-B aptamer C*AATTGGGCCCGTCCGTATGGTGGG*T “*” indicates the position of phosphorothioate (PS) modification. doi:10.1371/journal.pone.0050964.tKd 0.5060.32 nM 0.5660.44 nMAntiproliferative Activity of Aptamer on CancerFigure 2. SPR sensorgrams demonstrating interaction of PS-modified SL2-B aptamer with immobilized VEGF165 and VEGF121 protein at same concentration. Point A to B corresponds to association phase and point B to C corresponds to the dissociation phase in both the sensorgrams. Shown here is PS-modified SL2-B aptamer binding with VEGF165 protein (Kd = 0.5660.44 nM) and VEGF121 protein (Kd = 1761.24 mM) at 80 nM aptamer concentration. doi:10.1371/journal.pone.0050964.gaptamer sequence at 15 mM aptamer concentration. Same hypoxia conditions were maintained as for the antiproliferative activity assay. After 3 days of aptamer treatment, the cells were trypsinized, incubated with anti-human Jagged-1 fluorescein antibody for 1 hour, re-suspended in PBS buffer and analyzed immediately using a Beckman-Couter CyAn ADP flow cytometer by analyzing 15,000 events and relative fluorescence was determined using SUMMIT V 4.3.02 software.bands were developed with west pico chemiluminescence substrate and visualized on XPress CL blue ray film. Optical densities of bands were measured on 1531364 a GS800 densitometer and band intensities were analyzed with Quantity One image analysis software (Biorad, USA).Statistical AnalysisData are presented as mean 6 SD. A p-value ,0.05 was considered statistically significant using student’s t-test.Western Blot AnalysisThe sequence specific effect of PS-modified SL2-B aptamer on Jagged-1 protein expression in Hep G2 cells was analyzed using western blotting. Same experimental conditions were maintained as for the flow cytometry. After 3 days of aptamer treatment, the cell medium was removed and the cells were washed once in cold 16PBS. 500 ml of the complete lysis buffer was added to each 6well and the cells were scrapped with a cell scrapper and collected into microcentrifuge tubes. The extracted proteins were resolved on an SDS-PAGE gel and transferred onto a PVDF membrane via wet transfer. Membranes were blocked in 5 non-fat milk and washed in tris-buffered saline with 1 tween. Subsequently, membranes were incubated with primary antibody (Jagged-1 rabbit monoclonal and purified mouse anti-calnexin antibody) and then with corresponding secondary antibody (goat anti-rabbit and anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (HRP)) with 3 washing steps in between. The proteinResults and Discussion Binding Analysis of PS-modified SL2-B Aptamer and VEGF Complex by Surface Plasmon Resonance (SPR)As reported previously in our study, the unmodified SL2-B aptamer displayed a Kd = 0.5 nM to heparin binding domain (HBD) of VEGF165 protein determined via SPR technique (Table 1) [37]. The unmodified aptamer,.