Figure 4: Jag-1 rescues Notch3-induced endothelial cell death.

(a) HUVEC were stained with the CellTRacker green CMFDA. Cleaved-caspase-3 staining of HUVEC co-cultured with 1) LLC1 expressing or not Jag-1 (LLC1-Jag-1 and LLC1-Control, respectively) or 2) H358 cells transfected with a siRNA control (sicontrol) or a siRNA targeting Jag-1 (siJag-1). Co-cultures were maintained in matrigel for 9 h before being fixed and stained. Images are representative of three independent experiments, each performed in triplicate. (b) Quantification of HUVEC networks presented in a (n=3, means±s.d., t-test was applied). Jag-1 expression was verified on western blot before the cells were added to HUVEC in Matrigel. (c) Immunohistochemistry staining and quantification of CD31 on tumour section from tumours obtained from LLC1 cells or LLC1 cells overexpressing Jag-1 (n=5 tumours, quantification on 4 images/tumours, unpaired t-test). (d) Co-culture of HUVEC electroporated (Notch3) or not (Control) with Notch3 and LLC1 cells transfected (LLC1-Jag-1) or not (LLC1 control) with Jag-1 were stained with Annexin V APC and studied by flow cytometry. HUVEC were gated (M1) according to FL1 staining (cellTracker green CMFDA), and Annexin V (FL4)-positive cells were quantified among HUVEC. Number of Annexin V positive HUVEC cells is specified in each condition. (e) Tumour/normal tissue ratio of Jag-1 mRNA in patients with non-small-cell lung adenocarcinomas from the GSE7670 data set and Correlation between Jag-1 and Pecam-1 expression in patients overexpressing Jag-1 in the GSE7670 data set.