Notch signaling induced cellular extensions and VEGFR-1 expression in HUVEC. HUVEC were transduced with either Ad-LacZ or Ad-N1IC (MOI 40) and seeded on collagen type I gel for five days. Representative images from each cell culture are shown (10× magnification). (A) Ad-LacZ HUVEC did not form extensions, (B) while Ad-N1IC HUVEC underwent morphological changes as seen by the sprouting of extensions into the underlying matrix. (C) Morphological differentiation of Ad-N1IC HUVEC was not affected by the addition of 1 μM PD166866 (FGFR inhibitor), or (D) 1 μM ZD1893 (EGFR inhibitor). (E) The addition of 0.5 μM SU5416 (a VEGFR inhibitor), suppressed the morphological differentiation of Ad-N1IC HUVEC. (F) Quantification of the effect of the different tyrosine kinase inhibitors on Notch-induced cellular extensions and cell number. (G) Quantification of the effect of increasing amounts of the VEGFR inhibitor (SU5416) on Notch-induced cellular extensions and cell number. For morphogenesis assays, HUVEC with sprouting extensions per 10× microscopy field were counted, for five separate fields. Data is representative of the mean plus or minus SEM of three separate experiments, relative to control. Cell number was determined as percent of control using colorimetric cell proliferation kit. (H) RT-PCR of RNA isolated from mock (X) or N1IC-expressing retrovirally transduced HUVEC lines for VEGFR-1 (30 cycles), VEGFR-2 (30 cycles) and β-actin (25 cycles) (I) Western blot of total cell lysate from Ad-LacZ or Ad-N1IC transduced HUVEC to detect VEGFR-1 protein expression. Detection of α-tubulin was used as a loading control.