Fig 2: RND1 is the most highly regulated of the novel Notch targets in multiple contexts. (A) 12 genes were significantly regulated under all in vitro and in vivo RNA-seq screening conditions. Genes in purple are established Notch targets. (B) qPCR confirms that 7 of the 8 novel genes are regulated by DLL4 TLA in HUVEC and HREC, with RND1 showing the highest fold induction and the strongest inhibition by CpE. Black stars indicate significant induction by DLL4, red stars indicate significant inhibition of induction by CpE. (C) All novel targets are induced by EGTA; the most strongly induced gene is RND1 (blue line). (D) EGTA induction (blue or black lines) and CpE inhibition of EGTA (red lines) of the 8 novel putative endothelial Notch targets in HREC. Black stars indicate significant induction, red stars indicate significant inhibition of induction. The y axes are scaled for each gene to visualize induction of different magnitudes. (E) RND1, HIC1, and RGS3 remain induced after 48 h of DLL4 overexpression, while the other novel targets have transient expression that has returned to baseline after 48 h. (F) JAG1 TLA induces expression of most novel endothelial Notch targets at lower levels, suggesting that RND1 is a target of both DLL4 and JAG1 signaling. (G) qPCR of endothelial transcripts immunoprecipitated from the P8 mouse brain using an endothelial-specific RiboTag allele. Input = bulk brain homogenate, IP = immunoprecipitated endothelial mRNA.

Fig 3: Role of DNA methylation, TP53 and CTCF in regulation of DLL4 gene expressionA. silenced DLL4 by DNA methylation in MDA231 cell line is reactivated by inhibitor of DNA methylation 5'-aza-dC. I) DLL 4 expression level in MDA231 cell line is compared with HS27 and MCF7 by RT-PCR assay. II) Reactivation of silenced DLL4 gene in MDA231 by the DNA methylation inhibitor, 5-aza-dC. III) Methylation status of the DLL4 promoter in MDA231 cell lines treated with 5-aza-dC and detected by MS-PCR. IV) Phase-contrast photomicrographs showing morphologic changes in MDA231 cells treated with 2, 5µM, 10µM and 25µM of 5-aza-dC for 3 days, compared with untreated control cells maintained for 3 days. B. The effect of DNA methylation on interaction between CTCF and DLL4 promoter by ChIP assay C. Reactivation of DLL4 gene expression in LFS cell line, 3335, by CTCF and TP53 siRNA treatment. D. A schematic representation of presumable mechanism of regulation of DLL4 gene expression.

Fig 4: DNA methylation pattern of DLL4 gene promoter in LFS, breast and brain cancer cell linesA. Schematic representation of DLL4 promoter and CpG islands; B. Methylation status of the DLL4 promoter in LFS cell lines and cancer cell lines as detected by MS-PCR; C. Methylation status of the DLL4 promoter in human normal tissue and tumor tissue samples including colon, stomach and lung. D. Marked Reactivation of DLL4 gene expression in MCF7 but neither in the two DLL4-negative LFS cell lines nor in the normal cell line, HS27 by the DNA methylation inhibitor, 5-aza-dC.

Fig 5: MPDZ recruits DLL1 and DLL4 to Nectin-2.(A) DLL1 and DLL4 full length and such lacking the PDZ-binding site (?PDZ) constructs containing a mCherry tag were expressed in HUVEC. Cells were stained with antibodies against DLL1 or DLL4. DLL1 and DLL4 surface expression of mCherry positive cells was analyzed by flow cytometry. n = 3; *, p<0.05; **, p<0.01 unpaired Student’s t-test. (B) Endothelial cells were isolated from Mpdz+/+ embryos and Mpdz-/- littermates at embryonic day E11.5. Cells were purified by CD31 magnetic dynabeads and stained with anti-CD34 and anti-Dll4 antibodies for flow cytometric analysis. n = 4; *, p<0.05; unpaired Student’s t-test. (C, D) HEK293T control cells (293T sh-ctrl) as well as MPDZ-silenced HEK293T cells (293T sh-MPDZ) were transfected with Nectin-2 and HA-tagged DLL1 or Flag-tagged DLL4. For immunoprecipitation a Nectin-2 antibody was used and HA-tagged DLL1, Flag-tagged DLL4 and MPDZ were detected by western Blotting. Input, 10% of immunoprecipitate. IB, Immunoblot; IP, Immunoprecipitation; neg.ctrl., negative control.10.7554/eLife.32860.010Figure 3—source data 1.Source data of FACS analysis related to Figure 3A and B.