Fig 1: Notch directly regulates transcription of ERAD genes.(A) Quantification of surface NOTCH1 levels in different thymocyte subsets from wild-type mice. MFI, mean fluorescence intensity. n = four mice. (B) Quantitative RT–PCR analysis of ERAD genes (Sel1l, Hrd1, Os9, Edem1) expression in EL4 cells after stimulation with 5 µg/ml Delta ligand 4 (DLL4) for 24 hr. Data are presented relative to Actb. n = 3. (C) Western blot analysis of SEL1L level in EL4 cells after stimulation with Delta ligand 4 (DLL4) for 12 hr. ß-ACTIN was used as loading control. The original western blot images are provided in Figure 4—source data 1. (D) Quantitative RT–PCR analysis of ERAD genes (Sel1l, Hrd1, Os9, Edem1) expression in primary DN3 thymocytes treated with 2 µM ?-secretase inhibitor DAPT for 5 hr. Data are presented relative to Actb. n = 3. (E) Conserved RBP-J binding motif (Red) within the promoters of Sel1l and Hrd1. Alignment of the Sel1l (Upper) or Hrd1 (lower) promoter from genomic sequence from human, mouse, and rat. The numbering corresponds to the mouse sequence and is relative to the transcription start site (TSS). Mutations of the RBP-J-binding motifs within Sel1l or Hrd1 promoter luciferase reporters (as in L, M) are shown. (F–I). Upper: Schematic diagram of the ChIP primer (P1–P3) locations across the Sel1l (F) Hrd1, (G) Edem1, (H) or Os9 (I) promoter regions. TSS: transcription start site. Lower: Chromatin extracts from EL4 cells treated with PBS or 5 µg/ml DLL4 for 24 hr were subjected to ChIP using anti-RBP-J antibody, anti-NICD antibody, or normal IgG. Genomic regions of Sel1l (F), Hrd1 (G), Edem1 (H), or Os9 (I) promoter (as in left panel) were tested for enrichment of RBP-J, NICD or IgG. Data are shown as percentage of input. (J) Sel1l or Hrd1 promoter luciferase reporter was co-transfected with empty vector or different doses of NICD into HEK293T cells, and luciferase activity was measured 36 hr after transfection. pGL3 basic was used as control. (K and L) Wild-type or mutant (RBP-J motif mutations, as shown in E) Sel1l (K) or Hrd1 (L) promoter luciferase reporter was transfected into EL4 cells which were treated with PBS or 5 µg/ml DLL4 for 24 hr before harvest. Luciferase activity was measured 36 hr after transfection. All luciferase data are presented relative to Renilla readings. Data are shown as mean ± s.d. Two-tailed Student’s t-tests (A, B, D, F-I, K, L) or one-way ANOVA with Bonferroni test (J) were used to calculate p values. n.s., not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.Figure 4—source data 1.Original western blot images shown in Figure 4.Figure 4—source data 2.Excel file containing numerical values shown in Figure 4.
Fig 2: Notch1 modulates RBC‐induced hematopoiesis via Hes1. (A) Notch1 expression levels in HSCs after culture with or without RBCs for 1 day (n = 4 per group). (B–D) Flow cytometric analysis of LSK cells after culture of 500 HSCs with or without 1.0 × 107 RBCs for 1 day. (B) Histogram of LSK cells. (C) The percentages of Notch1+ LSK cells among LSK cells are shown (n = 3 per group). (D) Median fluorescence intensity (MFI) (n = 3 per group). (E) Hes1 expression levels in HSCs after culture of 500 HSCs with 1.0 × 107 RBCs and solvent control (DMSO) or 10 μM DAPT for 1 day (n = 3 per group). (F) Analysis of progenitor cells after culture of 500 HSCs with 1.0 × 107 RBCs and DMSO or 10 μM DAPT for 4 days. Numbers of WBCs, Lin− cells, LSK cells, and CD48− CD150+ LSK cells are shown (n = 3 per group). All data are presented as mean ± SD. P‐values are shown in the figures.
Fig 3: The O-Glc glycan is extended to form a 3’-sialyllactose-like structure. (A) The standard 3′-sialyllactose (Neu5Acα2-3Galβ1-4Glc) was permethylated following reductive β-elimination with sodium borodeuteride (NaBD4), resulting in a mass shift of +1 Da (m/z 839 in MS1). After β-elimination with sodium borohydride (NaBH4), the trisaccharide released from trypsin-digested glycopeptides, 366-TGLLCHLNDACIANPCNEGSNCDTNPVNGK-395, containing the EGF10 O-Glc site from NOTCH1 EGF6-10 was detected as its permethylated form (m/z 838 in MS1). For NSI-MSn analyses, permethylated trisaccharides were infused into a mass spectrometer in the presence of lithium ions, inducing linkage-specific sugar cross-ring cleavages (Right), as previously reported. Arrows indicate sugars fragmented in MS4 analyses (Right). The MS4 spectra (MS4 838@463@211) of the released trisaccharide correspond to those of the standard 3′-sialyllactose (Neu5Acα2-3Galβ1-4Glc) (Left). Both spectra yields signature fragments such as 3,5AGal at m/z 109 (blue), 1,3XGal at m/z 137 (yellow), and 2,3XGal at m/z 181 (green) identifying the presence of a 3-substituted internal hexose residue (33). Sugars that are cleaved off through neutral loss are shown as dashed gray symbols. (B–E) Glycoproteomics analysis of mouse NOTCH1 EGF10. MS spectra of mouse NOTCH1 EGF10 (glyco)peptides (TYEAFNDACISNPCNEGSNCDTNPVNGKAICTCPSGYTGPACSRGGPEQKLISEEDLNSAVDHHHHHH) with or without glycosidase treatment. B: Untreated control. C: α2-3-Neuraminidase treatment. D: β1-4-Galactosidase treatment. E: Treatment with both α2-3-Neuraminidase and β1-4-Galactosidase. Black bar: peptide; Blue circle: glucose; orange star: xylose; yellow circle: galactose; purple diamond: Neu5Ac. Theoretical m/z values of EGF10 peptides are as follows, naked: 1450.2169; Glc: 1482.7544, Xyl-Glc: 1509.1628; Xyl-Xyl-Glc: 1535.5712; Hex-Glc: 1515.1649; Neu5Ac-Hex-Glc: 1573.3840. The charge states of all peaks are +5.
Fig 4: Differential O-Glc elongation on NOTCH1 EGF10 regulates NOTCH1 signaling. (A–C) HEK293T cells overexpressing full-length Notch ligands DLL1 (A), DLL4 (B), or JAG1 (C) were incubated with mouse NOTCH1 EGF8-12 (0 to 5 ng/µL). Circles: mouse NOTCH1 EGF8-12, produced in POGLUT1-overexpressing WT HEK293T cells, containing both galactose- and xylose-elongated O-Glc glycans; squares: mouse NOTCH1 EGF8-12, produced in POGLUT1- and GXYLT1-overexpressing B4GALT1 KO cells, lacking galactose-elongated O-Glc glycans; triangles: mouse NOTCH1 EGF8-12, produced in POGLUT1- and B4GALT1-overexpressing GXYLT1- and GXYLT2-DKO cells, lacking xylose-elongated O-Glc glycans. All experiments were performed in triplicate and relative mean fluorescence intensity is shown on the y-axis. The intensity of mouse NOTCH1 EGF8-12 containing both galactose- and xylose-elongated O-Glc glycans was set as 1.0. The glycan composition of each NOTCH1 protein is shown in SI Appendix, Fig. S6. (D–H) RT-qPCR analysis of HES5 gene expression. Empty vector, WT, or mutant full-length NOTCH1-overexpressing cells were seeded on ligand-coated plates. D: DLL1-Fc stimulation (N = 3). E: DLL4-Fc stimulation (N = 3). F: JAG1-Fc stimulation (N = 3). The Ct value of HES5 was normalized by the GAPDH Ct value. Negative control IgG-Fc-stimulated cells were set as 1.0. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. Error bars represent STDEV. (G and H) Luciferase reporter assay. Empty vector, WT, or mutant full-length NOTCH1-overexpressing cells were seeded on ligand-coated plates. G: DLL1 Myc-His stimulation (N = 3). H: DLL4 Myc-His stimulation (N = 3). The relative luciferase unit of negative control IgG-Fc-stimulated cells was set as 1.0. Statistical analysis was performed using one-way ANOVA followed by Tukey’s test. Error bars represent STDEV.
Fig 5: NOTCH1 A396Y/F mutants promote T cell differentiation. (A–D) Three days after retrovirus introduction, LPs were transferred onto A: OP9-DLL1 or C: OP9-DLL4 stromal cells and cocultured for 4 d. CFP+hNGFR+ retrovirus-transduced cells were gated and analyzed for CD44 and CD25 expression. The relative percentage of CD25+ cells among CFP+hNGFR+ retrovirus-transduced cells after being cocultured with B: OP9-DLL1 or D: OP9-DLL4 is shown with STDEV. Data are representative of three independent experiments. Data represent mean values from three independent biological replicates. The data were analyzed by one-way ANOVA with Tukey’s multiple comparisons. (E) Summary of this study. The NOTCH1 activation is regulated by O-Glc glycans on EGF10. As summarized in Fig. 1A, O-Glc addition and xylosyl elongation on NOTCH1 are predicted to occur in nearly all EGF domains with consensus sequences in T cells. Exceptionally, in EGF10, the presence of Ala396 (C4-2) promotes 3’-sialyllactose elongation of O-Glc at Ser378, while suppressing xylosyl elongation. This regulation appears to occur in T cells. The amino acid sequence of EGF10 is partially shown. Oval: EGF repeats; blue oval: EGF repeats with O-Glc consensus sequence; blue circle: glucose; yellow circle: galactose; orange star: xylose; purple diamond: Neu5Ac; green arrow: upregulation; red arrow: downregulation.
Supplier Page from BioLegend for PE anti-mouse Notch 1