Figure 6.

Effect of cMet knockdown on Ad5-pIX-RFP-FF/NK2 infection.

Notes: (A) Confirmation of NK2 knockdown was determined in Hep 3B2.1-7 cells stably expressing shRNAs targeting cMet (Met/KD) or expressing non-specific shRNA (NT/Ctrl). Equal protein lysates were separated on 10% SDS-PAGE gels, transferred onto PVDF membranes, blocked, and probed with an anti-cMet monoclonal antibody. As a loading control, α-tubulin was used. Cell surface expression levels of cMet was determined in Hep 3B2.1-7 cells stably expressing shRNAs targeting cMet (Met/KD) or expressing non-specific shRNA (NT/Ctrl) by flow cytometry analysis and compared with cell surface expression levels of cMet in Hep 3B2.1-7 parental cells. The cells were incubated with PBS, an isotype control antibody or a cMet-specific antibody. Following incubation, the cells were analyzed by flow cytometry for the expression of cMet. In each experiment, 10,000 cells were analyzed for each sample. The analysis was repeated three independent times for each cell line. The data were represented as the percentage of gated cells positive for staining and expressed as the means of the three experiments ± SEM. The effect of cMet knockdown on Ad5-pIX-RFP-FF/NK2 binding was also determined. The Hep 3B2.1-7 cMet/KD and Hep 3B2.1-7 NT/Ctrl cell lines were infected with Ad5-pIX-RFP-FF/NK2 (B) or with the Ad5-pIX-RFP-wt/Fiber control (C) at increasing MOIs of 100 VP/cell, 1,000 VP/cell, and 2,500 VP/cell. After 60 minutes at 4°C, the cells were washed with ice-cold PBS and harvested. Total DNA was extracted, and Ad5 E4 DNA was measured by real-time PCR. The data were normalized to endogenous cyclophilin-specific DNA as an internal control. Each experimental condition was performed in triplicate, and the experiments were repeated two times. The results were expressed as means of the two experiments ± SEM. The two cell lines were compared using a two-tailed Student’s t-test. The differences were considered statistically significant (*) if P<0.05.

Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; PBS, phosphate-buffered saline; SEM, standard error of the mean; MOI, multiplicity of infection; VP, viral particles; PCR, polymerase chain reaction; Ad5, adenovirus serotype 5; NK2, a secreted truncated splicing variant that extends through the second kringle domain; shRNA, small hairpin RNA; NT/Ctrl, nontargeted control shRNA; Met/KD, cMet knockdown shRNA.