Figure 7.

The Notch ligand JAG1 is a direct target gene of Smad1/5 and transactivates Notch signaling in the neighboring cells. (A) Scatter plot representation of differentially regulated genes between HUVECs and PASMCs. Probes of genes with more than 2-fold change in expression relative to time 0 are plotted. If the genes are associated with Smad1/5 binding regions of HUVECs (green), PASMCs (red) or both (black), the plots are colored. Signal intensities of HUVECs treated with BMP-9 for 2 h is plotted on the X-axis and those of PASMCs treated with BMP-4 for 2 h is plotted on the Y-axis. (B) Visualization of JAG1 locus with the result of BMP-9 ChIP-seq. Red peaks represent ChIP regions (top panel). The conservation plots for mouse/human, frog/human and zebrafish/human are derived from VISTA genome browser (middle panel), which represents the sequence conservation between species. (C) Induction of JAG1 after BMP-9 stimulation in HUVECs. HUVECs were starved overnight, stimulated with 1 ng/ml BMP-9 and/or 10 ng/ml TNF-α for 2 h and subjected to qRT–PCR analysis for JAG1. Values were normalized to the amount of housekeeping GAPDH mRNA. The data are the mean of triplicate values ± SD. (D) HUVECs were starved overnight and stimulated with 1 ng/ml BMP-9 for indicated time periods and subjected to immunoblot analysis to determine the JAG1 protein expression level. α-Tubulin was used as a loading control. (E) Immunocytochemistry of HUVECs treated with or without 1 ng/ml BMP-9 for 24 h. The cells were immunostained with anti-JAG1 antibody (green). Nuclei were labeled with TOTO-3 (blue). Scale bar, 100 µm. (F) Endothelial JAG1 induced by BMP-9 stimulation transactivates Notch signaling in neighboring cells. HeLa cells were transiently transfected with pGL4-12xCSL-luciferase reporter construct and co-cultured with HUVECs. Cells were treated with or without 5 ng/ml BMP-9 for 24 h and subjected to luciferase assay. The data are the mean of triplicate values ±SD.