Combined overexpression of 8 TFs robustly induces the arteriovenous fresh profile in HUVEC-C. Combined overexpression of 8 TFs significantly induces an arterial expression profile in cultured HUVECs. (A) Heatmap analysis of the arteriovenous fresh profile upon transduction of cultured HUVECs with Cherry control virus, lentivirus expressing 1 TF, or a combination of all 8 TF-expressing lentiviruses (ALL). The farther the condition is removed from the Cherry control condition (or the greener the color in the Cherry column for a certain condition), the better the induction of the arteriovenous fresh profile. Note that the gold standard, HEY2, certainly is not the best-performing individual TF. (B) Hierarchical clustering analysis of the arteriovenous fresh profile upon transduction of cultured HUVECs with Cherry control virus, lentivirus expressing 1 TF, or a combination of all 8 TF-expressing lentiviruses (ALL). Red color in the bar on the left represents an arterial gene, whereas the blue color represents a venous gene. Although almost all the genes are expressed at low levels (in green) in the Cherry control condition, each of the individual TFs, except for Aff3, upregulated a subset of (mostly arterial) genes in a largely complementary but in some cases overlapping fashion. As expected from the complementarity, the 8 TFs together induce the majority of the (arterial) genes. (C-D) Diagrams representing expression for classical arterial (C, left) or venous (C, right) or general endothelial (D) markers in cultured HUVECs transduced with Cherry control virus (white) or all 8 TF-expressing lentiviruses (ALL TFs) relative to Cherry control. *P < .05 vs Cherry control. (E) Pie diagram representing the proportion of arterial genes with expression levels < 2% (light pink) or ≥ 2% (light red) in cultured HUVECs transduced with all 8 TF-expressing lentiviruses (ALL TFs) relative to those in freshly isolated HUAECs (upper). Pie diagram representing, within the subset of arterial genes with ≥ 2% values, the proportion of genes with midrange (<25%; dark pink) or high-range (≥ 25%; dark red) expression (lower). (F) Western blot showing the validation of some genes being upregulated by TF overexpression. α-TUBULIN was used as loading control. D, proportional values for the density of the protein band; kDa represents expected protein size.