Fig. 6.

Disruption of neuropilin-1 (nrp1) gene expression in cultured mesenchymal cells impaired their migration. Parenchymal pulmonary mesenchymal cells were isolated from lungs of Nrp1F/F mice at postnatal day 10 (P10) or P12 and cultured. Cre recombinase was introduced using adenovirus 5 (Ad5) and the CMV promoter driving transcription of Cre recombinase (Ad5-CMV-Cre) or an empty cassette (Ad5-CMV-empty) at multiplicity of infection (MOI) of 10 or 25. A: Ad5-Cre significantly reduced NRP1 mRNA by 12 h and NRP1 protein by 24 and 48 h. Values are means ± SE; n = 3 separate cell isolations. *P < 0.01 (by unpaired t-test). B: density of NRP1 relative to density of β-tubulin (βTub) for that lane. For cultures that received Ad5-CMV-Cre, this ratio was expressed relative to that for the culture that was comparably transduced with Ad5-CMV-empty and is shown below the 4 lanes. Results are representative of 2 other similar experiments. C: fibroblasts were isolated from lungs of Nrp1F/F mice and adhered to Vitronectin-coated glass within 8-well sticky-Slide chambers. At 24 h after transduction with Ad5-CMV-Cre or Ad5-CMV-empty, lung fibroblasts (LF) were allowed to migrate to an intervening empty space in the presence or absence (control) of platelet-derived growth factor (PDGF)-A. Some wells received no virus. Cumulative number of cells that had entered the previously empty space is plotted at 1-h intervals. Values are means ± SE; n = 3 separate cell isolations from separate litters for each genotype. *P < 0.01 vs. Ad5-empty (medium containing PDGF-A) (by repeated-measures ANOVA).