Figure 5.

Analysis of Ad5-pIX-RFP-FF/NK2 binding specificity.

Notes: (A) Using Hep3B2.1 cells, binding of Ad5-pIX-RFP-FF/NK2 was compared with the control Ad5-pIX-RFP-wt/Fiber after HGF pretreatment. The cells were pretreated with increasing concentrations of HGF, followed by infection with either Ad5-pIX-RFP-FF/NK2 or the Ad5-pIX-RFP-wt/Fiber control at an MOI of 10 VP/cell. After 2 hours of incubation at 37°C, the cells were washed and incubated for an additional 48 hours at 37°C. Afterward, the cells were washed, harvested, and the RFP reporter gene expression was measured by flow cytometry. All the data were normalized to HGF-untreated cells. Each experimental condition was performed in triplicate, and the experiments were repeated three times. The results were expressed as the mean of the three experiments ± SEM. Differences were considered statistically significant by Student’s t-test if P<0.05. (B) Using Hep3B2.1 cells, binding of Ad5-pIX-RFP-FF/NK2 was compared with the control Ad5-pIX-RFP-wt/Fiber after pretreatment with an anti-cMet antibody. The Hep3B2.1 cells were preincubated with increasing antibody concentrations for 30 minutes at 4°C. The cells were then washed, followed by infection with either Ad5-pIX-RFP-FF/NK2 or with Ad5-pIX-RFP-wt/Fiber for 30 minutes at 4°C using an MOI of 10 VP/cell. After incubation, the cells were harvested, and total DNA was extracted. The Ad5 E4 DNA content was measured by real-time PCR and plotted against anti-cMet antibody concentration. The data from antibody-treated cells were normalized to cells that were untreated with the anti-cMet antibody. Each experimental condition was performed in triplicate, and the experiments were repeated three times. The results were expressed as the mean of the three experiments ± SEM. The treatment results were compared using the Student’s t-test; the differences were considered statistically significant (*) if P<0.05.

Abbreviations: HGF, hepatocyte growth factor; MOI, multiplicity of infection; VP, viral particles; SEM, standard error of the mean; PCR, polymerase chain reaction; RFP, red fluorescent protein; Ad5, adenovirus serotype 5; NK2, a secreted truncated splicing variant that extends through the second kringle domain