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: NOTCH1 EGF10 is modified by O-Glc elongated by galactose and Neu5Ac. MS data of protease-digested NOTCH1 EGF10. (A) Summary of O-glucosylation on NOTCH1 (Top) (18, 25, 26). Summary of O-fucosylation and O-GlcNAcylation on NOTCH1 (Bottom) (16, 19). The most elongated glycan structures at each glycosylation site are shown. Minor glycoforms at certain EGF repeats are shown with dashed outlines and striped patterns. Blue circle: glucose; orange star: xylose; white circle: hexose; purple diamond: Neu5Ac; white oval: EGF; blue oval: EGF with O-Glc consensus sequence; blue and white striped oval: EGF modified with hexosylated O-Glc; red arrow: EGF with O-Fuc consensus sequence; green arrow: EGF with O-GlcNAc consensus sequence. (B) Tryptic digest of mouse NOTCH1 EGF1-12. Left: MS/MS spectrum of peptide, 366-TGLLCHLNDACISNPCNEGSNCDTNPVNGK-395; Middle: EICs of each (glyco)peptide; Right: Stoichiometry of each glycoform shown as a bar graph (N = 3). (C) V8 digest of mouse NOTCH1 EGF1-12. Left: MS/MS spectrum of peptide, 375-ACISNPCNE-383; Middle: EICs of each (glyco)peptide; Right: Stoichiometry of each glycoform shown as a bar graph (N = 3). In the Left panels, representative peaks are annotated. The color coding of EICs and bar graphs is shown at the Bottom. Black: non-O-glucosylated peptide; blue: peptide with Glc monosaccharide; yellow: peptide with Xyl-Glc disaccharide; orange: peptide with Xyl-Xyl-Glc trisaccharide; red: peptide with Hex-Glc disaccharide; purple: peptide with Neu5Ac-Hex-Glc trisaccharide. Error bars represent the SD.
Fig 4: 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 5: B4GALT1 and ST3GAL4 are involved in the O-Glc glycan biosynthesis. Mouse NOTCH1 EGF6-10 was digested by trypsin and analyzed by MS. For the analysis with B4GALT5- and B4GALT6-DKO cells, mouse NOTCH1 EGF1-12 was digested by trypsin. EICs of (glyco)peptides, 366-TGLLCHLNDACISNPCNEGSNCDTNPVNGK-395, are shown. (A) WT HEK293 cells. The red arrow indicates galactose-extended O-Glc glycan and the purple arrow indicates galactose- and Neu5Ac-extended O-Glc glycan. (B) Multiple B4GALT gene–deficient HEK293 cells. (C) Single B4GALT gene–deficient HEK293 cells. (D) Multiple ST3GAL and/or ST6GAL gene–deficient cells. (E) Single ST3GAL gene–deficient cells. The color coding of EICs is shown in the Bottom Right. Black: non-O-glucosylated peptide; blue: peptide with Glc monosaccharide; yellow: peptide with Xyl-Glc disaccharide; orange: peptide with Xyl-Xyl-Glc trisaccharide; red: peptide with Gal-Glc disaccharide; purple: peptide with Neu5Ac-Gal-Glc trisaccharide.
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