Reactivation of Notch signaling only partially restores arterial gene expression in HUAEC-C. Canonical Notch signaling is lost upon culture and its reactivation only partially restores the arterial signature in HUAECs. (A-B) Schematic diagrams showing 4 numbered consecutive steps during normal canonical Notch signaling in vivo (A) or under several experimental conditions in vitro (B), ie, in the presence of DAPT (left), siRNA against RBPJ (siRBPJ; middle), or Delta-like (DLL)4-Fc anchored to the cell culture dish (right). (C) Diagram representing expression (±SEM; n = 4) of several Notch pathway members and downstream genes in cultured HUAECs treated for 72 hours with DAPT (black) or dimethlysulfoxide (DMSO) (white) relative to DMSO. *P < .05 vs DMSO. (D) Diagram representing expression (±SEM; n = 4) of the same gene panel in cultured HUAECs treated for 6 days with siRBPJ (black) or nonsilencing siRNA (siNS; white) relative to siNS. *P < .05 vs siNS. (E) Diagram representing expression (±SEM; n = 4) of the same gene panel in cultured HUAECs treated for 72 hours with BSA/DAPT (light gray), BSA/DMSO (white), DLL4-Fc/DMSO (black), or DLL4-Fc/DAPT (dark gray) relative to BSA/DMSO. *P < .05 vs BSA/DMSO or DLL4-Fc/DMSO. (F) Diagram representing expression of all arterial (left) and venous (right) genes of the arteriovenous fresh profile in cultured HUAECs exposed for 72 hours to DLL4-Fc relative to BSA (red dotted line indicates expression in BSA-treated cells). Genes with no change in expression are in white, those that are upregulated are in black, and those that are downregulated are in gray. *P < .05 vs BSA. d, day.