Figure 2.

Activation of αvβ3 integrin signaling in normal HTM cells increases fibronectin matrix assembly. (A) Confluent monolayers of HTM cells were left untreated or treated for 12 days with vehicle (0.1% EtOH) or 500 nM DEX alone or in the presence of increasing concentrations of the CGT peptide. Cell layers were then extracted with 1% DOC and processed for OCW analysis. There was no difference in the levels of fibronectin fibrils between untreated cells and cells treated with EtOH alone. Cells treated with EtOH and 100 μM CGT showed significant increases in DOC-insoluble fibronectin levels compared to control cells treated with EtOH alone (P < 0.005). Although fibronectin levels were higher in cells treated with EtOH plus 50 or 200 μM CGT, the increases were not statistically significant. DEX treatment alone also significantly increased DOC-insoluble fibronectin levels relative to cells treated with EtOH alone (P < 0.002). In the presence of all three CGT concentrations, DOC-insoluble fibronectin fibril levels were significantly increased further in DEX-treated cells relative to DEX treatment alone (50 μM CGT, P < 0.01; 100 and 200 μM CGT, P < 0.002). The data reported are from one experiment that was performed twice with similar results. (B) Immunofluorescence microscopy images of HTM cells transduced with Ad5-EV or Ad5-WTβ3-mCherry or Ad5-CAβ3-mCherry viral vectors. Subconfluent cells were transduced at an MOI of 100 and processed for immunofluorescence 12 days post transduction. Scale bar: 500 μm. (C) OCW analysis of 1% DOC-extracted HTM cell monolayers transduced with Ad5 vectors described in (B). Twelve days post transduction, Ad5-CAβ3–transduced cells significantly increased DOC-insoluble fibronectin fibrils relative to both Ad5-EV–transduced cells (P < 0.003) and Ad5-WTβ3–transduced cells (P < 0.03). The data reported are from one experiment that was performed twice with similar results.