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. 2020 Jan 23;11:460. doi: 10.1038/s41467-019-14076-3

Fig. 5. ZEB1 depletion epigenetically reduces Dll4 and Notch1 expressions in bone ECs.

Fig. 5

a Schematic representation of reporter constructs of wild-type (WT) and mutated (MUT) murine Dll4 and Notch1 promoters. b Luciferase reporter analysis of cultured control and ZEB1-deleted bone ECs that were electroporated with WT or MUT Dll4 and Notch1 promoter reporter constructs (n = 3 independent experiments). c Luciferase reporter analysis of cultured bone ECs that were co-electroporated with WT or MUT Dll4 and Notch1 promoter reporter constructs in combination with pcDNA3.1 or pcDNA3.1-HA-ZEB1 (n = 3 independent experiments). d, e ChIP-PCR analysis of interactions of ZEB1-Dll4 promoter and ZEB1-Notch1 promoter within the proximal and distal regions in cultured bone ECs. Agarose gel images as shown are from three independent experiments (d) and promoter occupancy relative to corresponding input was quantified (e; n = 3 independent experiments). prox. proximal, dist. distal. f ChIP-qPCR for analyzing the enrichments of H3K4Ac, H3K14Ac, H3K18Ac, and H3K27me3 on Dll4 and Notch1 promoters in control and ZEB1-deleted bone ECs (n = 3 independent experiments). g Immunoprecipitation (IP) analysis of the physical interaction between ZEB1 and CBP or p300 in nuclear extracts of bone ECs. Images as shown are from three independent experiments. h Sequential ChIP-PCR analysis confirming the co-occupancy of ZEB1 and CBP or p300 on Dll4 and Notch1 promoters in bone ECs. Images as shown are from three independent experiments. i, j Luciferase reporter assays for analyzing Dll4 and Notch1 promoter activity in HEK293T cells that were co-transfected with the indicated constructs (i). Immunoblot analysis confirming ectopic expression of HA tagged ZEB1, p300, and CBP in nuclear extracts of HEK293T cells (j). Images as shown are from three independent experiments. Arrow marks specific bands with the expected molecular weights, respectively. All data are represented as mean ± SD. **P < 0.01, *P < 0.05; NS not significant. Differences are tested using one-way ANOVA with Tukey’s post hoc test (b, c, i) and unpaired two-tailed Student’s t-test (e, f). The source data are provided as a Source Data file. Unprocessed original scans of blots are shown in Source Data file.