Fig. 4

Fig. 4

a Model for signaling in activated versus quiescent endothelium. b Diagram illustrating tissues isolated from mice and their use. c Quantification of vascular phospho-ERK1/2 (black bars) and phospho-AKT (gray bars) levels in immunostained liver or tumor tissue from LLC-allografted control animals (n = 8). d ELISA measurement of PIP2 levels in lung or tumor endothelial cells from LLC-allografted control animals. Data is the average of three experimental replicates. e–nCds2 vMO-treated, LLC-allografted mice. Quantification of PIP2 levels by ELISA in tumor (e) or lung (j) endothelial cells isolated from the same LLC-allografted animals. Data per treatment condition is graphed as the average of three experimental replicates. The ELISA measurements are normalized to whole cell tubulin lysates collected at the start of the lipid isolation procedure. Quantification of vascular phospho-ERK1/2 (f, k), phospho-AKT (g, l) and CDS2 (h, m) levels from immunostained sections of tumor (f–h) or liver (k–m) tissue collected from the same LLC-allografted animals. For all immunostaining quantitation experiments: three images per individual tumor or liver were acquired and signal intensity measured for all groups. A minimum of two slide sections from each tumor or liver (taken from sections at least 10 slices apart) were utilized for independent analysis. Tumors, n = 10; Livers, n = 5. i, n Representative images of tumor (i) or liver (n) sections from control or Cds2 vMO-treated mice, double immunostained with phospho-ERK1/2 antibody (green images) and PECAM antibody (red images). o–t L-690,488-treated, LLC-allografted mice. Quantification of vascular phospho-ERK1/2 (o, r) and phospho-AKT (p, s) levels from immunostained sections of tumor (o, p) or liver (r, s) tissue collected from the same LLC-allografted animals. q, t Representative images of tumor (q) or liver (t) sections from control (DMSO) or L-690,488-treated mice, double-immunostained with phospho-ERK1/2 antibody (green images) and PECAM antibody (red images). Control tumors, n = 8; L-690,488 tumors, n = 16; control livers, n = 4; L-690,488 livers, n = 8 p ≤  0.05, error bars ± SEM. Star indicates significance from control (t-test). Bars = 200 μm. Box plots are graphed showing the median versus the first and third quartiles of the data (the middle, top, and bottom lines of the box, respectively). The whiskers demonstrate the spread of data within 1.5x above and below the interquartile range. All data points are shown as individual dots, with outliers shown above or below the whiskers.