Fig. 4. Endothelial ZEB1 inactivation downregulates Notch activity.

a RT-qPCR analysis of Zeb1, Zeb2, Dll4, Notch1, Notch2, Notch3, Notch4, Hes1, Hes5, Hey1, Heyl, and Efnb2 in FACS-sorted bone ECs of 3-week-old Zeb1^WT and Zeb1^ΔEC mice (n = 3 independent experiments). b, c Representative confocal images of Dll4 (left panels) and Notch1 (right panels) immunostaining in the tibia of 3-week-old Zeb1^ΔEC mice (b; n = 5) and Zeb1^iΔEC mice (c; n = 5), and their corresponding littermate controls (n = 5, each). Scale bar, 60 μm. d Quantification of Dll4 (left panels) and Notch1 (right panels) staining levels in EMCN⁺ bone ECs of 3-week-old Zeb1^WT and Zeb1^ΔEC mice as described in b (n = 5 independent experiments). e Quantification of Dll4 (left panels) and Notch1 (right panels) staining levels in EMCN⁺ bone ECs of 3-week-old Zeb1^WT and Zeb1^iΔEC mice as described in c (n = 5 independent experiments). f Representative phase contrast (top panel) and confocal (CD31 immunostaining; bottom panel) images of magnetic activated cell sorting (MACS)-sorted bone ECs. Scale bar, 50 μm. g RT-qPCR analysis of Zeb1 and the indicated Notch pathway components in the in vitro cultured control (i.e., adeno-βGal-infected) and ZEB1-deleted (adeno-Cre-infected) bone ECs (n = 3 independent experiments). h Immunoblot analysis of ZEB1, Dll4, Notch1, and NICD1 in control and ZEB1-deleted bone ECs as described in g. Representative blots as shown are from three independent experiments. i Representative confocal images of control and ZEB1-deleted bone ECs with immunostaining of ZEB1, Dll4, Notch1, and NICD1. Scale bar, 20 μm. Images as shown are from three independent experiments. All data are represented as mean ± SD. **P < 0.01, *P < 0.05. Differences are tested using one-way ANOVA with Tukey’s post hoc test (a, g) and unpaired two-tailed Student’s t-test (d, e). The source data are provided as a Source Data file. Unprocessed original scans of blots are shown in Source Data file.