Effects of the Src family kinase inhibitor, PP2, on basal and 007-induced cell spreading.

<p>Cells were trypsinised, rolled for 1 hour without or with 20 µM PP2 pre-treatment and plated on fibronectin with or without 007. Adhesion of A549-Epac1 cells after 30 minutes was determined by alkaline phosphatase activity (A). PP2-treated A549-Epac1-GFP-Lifeact cells were plated on fibronectin and imaged over time using the 63×objective. Shown are representative images (B) and quantification of the mean spread area ± the standard error of the means (C) of 16 cells plated without 007 and 15 cells with 100 µM 007 from three separate spreading experiments. Spread area was standardised to the average area of −007 cells shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050072#pone-0050072-g001" target="_blank">Figure 1B</a> at the first frame of imaging (from assays performed independently, but under comparable experimental conditions). The dashed grey curve shows the spreading kinetics of 007-treated cells from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050072#pone-0050072-g001" target="_blank">Figure 1B</a>. The vertical line indicates the time that cells reached their maximum spread area. The spreading rate was calculated using the gradient (m), in spread area per hour, of the red line shown on the graph. In (D) and (E), A549-Epac1-GFP-Lifeact-expressing cells captured through the 20×objective spread in the presence or absence of 100 µM 007 for 2.5 hours before 20 µM PP2 was added. For quantification (E), the spread area was standardised to the average area of −007 cells at the first time point of imaging. In (F), human umbilical vein endothelial cells were plated on fibronectin for 1 hour in the presence and absence of 100 µM 007 and 20 µM PP2, before being fixed and stained. At least 30 cells per condition were quantified and standardised to the mean area of cells spreading without 007 or PP2. In (A) and (F), graphs show the mean of 5 and 3 experiments, respectively ± the standard error of the means. <i>P</i> values were calculated by paired student’s t-tests.