Integrin α1 cytoplasmic tail mutants activate distinct signaling pathways in endothelial cells. (A) The cell populations were plated in serum-free medium on solidified collagen I + IV gels. Tubulogenesis was quantified 16 hours after plating as described in “Tubulogenesis.” Values are mean plus or minus SD of 4 independent experiments. Gels were viewed with a Nikon Diaphot inverted research microscope (Nikon, Tokyo, Japan) using a lens at 20×/0.2 Ph2 LD 0.4. Images were acquired using a Canon PowerShot S5 IS camera (Canon USA, Lake Success, NY) and were processed with Adobe Photoshop version 9.0 software (Adobe Systems, San Jose, CA). (B) The cell populations were plated in 96-well plates coated with 10 μg/mL collagen IV. Four hours later, the cells were incubated with serum-free medium containing ³H-thymidine (0.5 μCi/well) for a further 48 hours, and proliferation was then evaluated as described in “Cell proliferation.” Values are mean plus or minus SD of one representative experiment performed in quadruplicate. *Statistically significant differences (P < .05) between the α1KO cells and α1 mutant–expressing cells. (C) The cell populations were serum-starved for 24 hours and embedded in collagen I + IV gels for the time indicated. The gels were sonicated and run on an SDS-PAGE gel to detect levels of activated and total p38 MAPK, ERK, and Akt. Images are representative of 3 independent experiments. Vertical line(s) have been inserted to indicate a repositioned gel lane. (D) Phosphorylated and total kinase bands were quantified by densitometry analysis, and the phosphorylated signal was expressed as phosphorylated kinase/total kinase ratio. Values are the mean plus orf minus SD of 3 independent experiments. * indicates significant differences (P < .05) relative to α1KO cells. (E-G) The cell populations indicated were subjected to migration (E), tubulogenesis (F), and proliferation (G) assays on collagen IV or fibronectin in the presence or absence of 10 μM PD169316, 10 μM PD98059, or 5 μM LY294002. Values are the mean plus or minus SD of one representative experiment. Differences between untreated α1KO and α1 mutant–expressing cells (*) and inhibitor untreated versus treated α1 mutant–expressing cells (#) were significant with P < .05.