Extended Data Fig. 6: Regulation of MYC by DLL4–NOTCH signalling in vivo and in vitro.
From: Arterialization requires the timely suppression of cell growth
a, MYC–GFP fusion protein expression is upregulated in coronary vessels (ICAM2+) after inducing heterozygous Dll4 deletion. b, Comparison of MYC protein expression in wild-type and Rbpj mutant ERG+ ECs. c, Western blot analysis showing no major changes in MYC protein levels after stimulation of HUVECs with DLL4 ligands for 6 or 24 h, in the presence or absence of the Notch inhibitor DBZ. d, qRT–PCR analysis showing significant changes of Notch target genes (Dll4, Hey1, Hey2, Hes1), but not Myc and Odc1, after stimulation of HUVECs with DLL4 ligands for 8 h in the presence or absence of the Notch inhibitor DBZ. e–h, Western blot and qRT–PCR analysis showing that an even stronger activation of Notch signalling (for 24 h, e and f; and 48 h g and h) and its downstream target genes (HEY1, HEY2, NRARP and HES1) in HUVECs overexpressing Dox-inducible N1ICD-V5 has relatively minor or non-significant effects on MYC expression. ODC1 expression is 40% decreased. i, Whole-mount analysis of control and MYC mutant hearts showing that global MYC deletion compromises the development of coronary vessels and consequently arteries (CX40+). j, Sectional analysis of control and MYC mutant hearts showing a severe depletion of coronary vessels in the myocardium which causes a reduction in its thickness. k, Mosaic induction of Mycflox/flox Cdh5-creERT2 iSuRe-cre mice shows that MYC-null ECs (MbTomato-2A-Cre+) form well coronary arteries, in contrast to hearts with full induction of Myc deletion. Data shown as mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 100 μm. For statistics, see Supplementary Data 1. For western blot gel source data, see Supplementary Fig. 1. Western blot controls (tubulin) were run on separate gels, owing to overlapping size with MYC, as sample processing controls.
