Figure 2: Tie2-dependent signalling controls pericyte migration.

(a) Representative images of control BP (shCtr) and silenced for Tie2 (shTie2 I and shTie2 II) at the beginning (t=0 h) and end (t=6.5 h) of time-lapse microscopy during lateral-scratch wound assay. (b) Quantification of covered area by BP shTie2 I and shTie2 II in a lateral-scratch wound assay after 6.5 h normalized to BP shCtr. (c) Quantification of covered area by BP treated with supernatants of control (Ctr) or Ang2-overexpressing (pAng2) HUVEC in a lateral scratch wound assay after 6.5 h normalized to control. (d) Representative images of calpain 1 (CAPN1) staining of BP stimulated with recombinant human (rh) Ang2 for 1 h or left unstimulated (us). Nuclei were counterstained with Hoechst. (e) Quantification of calpain 1 activity measured by detection of fluorescent cleaved substrate in BP stimulated for 1 h with rhAng2 normalized to unstimulated (us) control (n=3). (f) Western blot analysis of phosphorylated FOXO3A (pFOXO3A) and total FOXO3A in BP stimulated with rhAng1 or rhAng2 for 30 min. Tubulin served as loading control. (g) Representative images showing FOXO3A localization in BP stimulated with rhAng1 or rhAng2 for 30 min. Cells were further stained with actin (cytoskeleton staining) and Hoechst (nucleus staining). (h) Human phospho-receptor tyrosine kinase array for EphA2, EphB2 and EphB4 ephrin receptors from control BP (shCtr) and BP silenced for Tie2 (siTie2 I and siTie2 II). Intensity is shown as fold of siCtr. Representative western blot and array images are cropped versions and original images can be found in Supplementary Figs 15 and 17. Scale bars: 250 μm (a), 50 μm (d,g). Data are shown as mean±s.d. Statistics were performed using one-way ANOVA (b), Mann–Whitney U test (c) and Student’s t-test (e). *P<0.05, ***P<0.001.