Fig 2: NOTCH1 mutations in CLL.A) Position of NOTCH1 mutations, superimposed on a cartoon showing the NOTCH1 protein. NRR, negative regulatory region; RAM, RAM domain; ANK, ankyrin repeat domain; TAD, transcriptional activation domain; PEST, degron domain. B) Representative confirmatory pyrosequencing results in CLL cases positive by deep sequencing for the NOTCH1 codon 2514 del(CT) mutation. Results for a case with wild type codon 2514 sequences is shown in the upper panel. The lower two panels are cases in which deep sequencing revealed high (54.1%) or low numbers of mutated reads (3.3%). Reverse-sequences of the wild type (WT) and mutant (MUT) del(CT) alleles are shown for reference. “E” corresponds to the addition of enzyme to the reaction chamber, while “S” corresponds to the addition of the substrate. In this sequencing by synthesis assay, nucleotides are added to the reaction chamber sequentially in the order shown. Nucleotide incorporation releases pyrophosphate, which catalyses a reaction in which the number of photons produced is proportional to the number of nucleotides incorporated. The codon 2514 del(CT) mutation causes the appearance of a new signal corresponding to the incorporation of 3 G residues.

Fig 3: Notch signaling is downregulated in aortic medial VSMCs in DTAAD patients.A) Immunofluorescence double staining showed that DLL1/4 in VSMCs of the aortic media was significantly decreased in both TAA and TAD tissues compared with control tissue. B) Notch1 in VSMCs of the aortic media was significantly decreased in both TAA and TAD tissues compared with control tissue (scale bar = 25 µm, insets 6.25 µm). C) NICD was rarely detected in vascular smooth muscle cells (VSMCs) of the aortic media in TAA and TAD tissues. D) Hes1 was rarely detected in VSMCs of the aortic media in TAA and TAD tissues (scale bar = 50 µm).

Fig 4: FGF signaling regulates Hey2 expression in a Notch1 dependent manner. (a) Notch1 is deleted efficiently in ROSA26CreERT2; Notch1fl/fl MEFs via 4-OHT treatment. (b) Q-PCR analysis shows that Hey2 mRNA level is increased with FGF2 stimulation in control MEF cell and decreased in Notch1 null MEF cell, while FGF2 stimulation doesn’t cause significant change in Notch1 null MEFs compared with control based on student’s t-test. (c and d) Notch1 is deleted efficiently in ROSA26CreERT2; Notch1fl/fl MEFs by 4-OHT treatment. p-Erk protein level decreased in Notch1 null MEFs. FGF2 stimulation increases p-Erk protein level in control MEFs but not in Notch1 null MEFs. FGF2 stimulation promoted Hey2 protein level in control MEFs but not Notch1 null MEFs. An unpaired, two-tailed student’s t-test was used for statistical comparison. The Representative pictures from three repeats were shown and the original pictures of all the blots are shown in Suppl. Figure 5.

Fig 5: 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 Zeb1WT and Zeb1?EC 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?EC mice (b; n = 5) and Zeb1i?EC 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 Zeb1WT and Zeb1?EC 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 Zeb1WT and Zeb1i?EC 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-ßGal-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.