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. 2023 Feb 9;14:731. doi: 10.1038/s41467-023-36409-z

Fig. 7. Endothelial Sp1/Sp3 negatively regulates Notch signaling to govern angiogenesis.

Fig. 7

A Western blot analysis of Notch1, Notch intracellular domain (NICD), DLL4, Sp1, and Sp3 protein levels in isolated retinal endothelial cells from CTR and dKO mice. B qPCR analysis of NOTCH1, HES1, HEY1, and DLL4 mRNA levels in isolated retinal endothelial cells from CTR and dKO mice. n = 6. C Representative microscopy images and quantification of immunofluorescence staining for Notch1 in isolectin-B4+ vessels of retinas from CTR and dKO mice. Scale bar: 30 μm. n = 5. D Representative microscopy images and quantification of immunofluorescence staining for phospho-VEGFR2 (Y1175) in isolectin-B4+ vessels of retinas from CTR and dKO mice. Scale bar: 30 μm. n = 5. E Retinal whole-mount staining of isolectin-B4 in P5 CTR and dKO mice, with or without the DAPT (γ-secretase inhibitor). Red arrowheads show tip cell sprouting and filopodia. Bottom, quantification of vascular/total retinal area, vasculature length, branch points per field, tip cell number per field, tip sprout number per field, and filopodia number per sprout. Scale bars: 500 (row 1), 100 (row 2), 50 (row 3), and 10 μm (row 4). n = 6. F Representative laser Doppler images of legs on days –1 (before surgery), 0 (immediately after surgery), 7, 14, 21, and 28. Bottom, quantification of blood flow recovery after hindlimb ischemia as determined by the ratio of foot perfusion between ischemic (left) and non-ischemic (right) legs in CTR and dKO mice with or without DAPT. n = 6. G IHC analysis of CD31+ staining (capillary density) in the ischemic gastrocnemius muscle. Right, quantification of CD31+ vessels per mm2 in CTR and dKO mice, with or without DAPT. Scale bar: 50 μm. n = 6. H Western blot analysis of phosphorylated VEGFR2 (Y1175, Y1059), VEGFR2, PLCγ (Y783), PLCγ, ERK1/2 (Thr202/Tyr204) and ERK1/2 in mithramycin or Ad-NICD treated HUVECs. Two-tailed Student’s unpaired t-test was used for B–D. One-way ANOVA followed by Bonferroni multiple-comparison analysis was employed for E and G. Two-way ANOVA followed by Bonferroni multiple-comparison analysis was performed for F. Data are presented as mean ± SEM. Source data are provided as a Source Data file.