Figure 1.
AdRac1N17 infection increased Rac1 protein levels, decreased Rac activity, and inhibited growth in MIAPaCa-2 cells. A. Cells transduced with 0-100 MOI AdRac1N17 demonstrated increases in Rac1 immunoreactivity with increasing viral titer. No difference was seen with AdBglII transfection compared with parental cells. For total Rac1 measurement, 30 μg cellular protein per well was used for western. All of the western blots were repeated to confirm the findings.
B. Representative western blot showing dose-dependent decreases in Rac1 activation in AdRac1N17-transduced MIAPaCa-2 cells. AdBglII served as a control. Cell lysates were immunoprecipitated with PAK-1 PBD-agarose. Associated GTP-bound Rac1 was determined by immunoblotting with anti-Rac1 antibody. 10 nM GTP was added to untreated cells as the positive control.
C. Overexpression of AdRac1N17 delayed MIAPaCa-2 cell growth. MIAPaCa-2 cells transduced with 0 100MOI AdRac1N17 or 100 MOI AdBglII demonstrated reductions in cell growth with AdRac1N17100 MOI. No significant changes were seen with AdBglII transduction compared with parental cells. Mean in vitro cell growth of control, AdRac1N17-or AdBglII-transduced MIAPaCa-2 cells are shown. Each point represents the mean values, n= 3. *P < 0.05 vs. 100 MOI AdBglII.
D. Plating efficiency was decreased with increased AdRac1N17 transduction in MIAPaCa-2 cells. MIAPaCa-2 cells were seeded at 5 × 105 cells / plate overnight until attached, transduced by AdRac1N17 or AdBglII for 24 hours, recovered in full media without adenovirus for another 24 hours, and were then trypsinized and plated for clonogenic survival. AdRac1N17 transduced cells decreased plating efficiency at 100 MOI. No significant changes were seen with AdBglII transfer compared with parental cells. Each point represents the mean values, n = 3. *P < 0.05 vs.100 MOI AdBglII.