Fig 2: EOGT promotes NOTCH1 binding to Delta ligands.(A) Diagram of predicted O-glycans on mouse NOTCH1: red triangle, O-fucose glycans; blue circle, O-glucose glycans; blue square, O-GlcNAc glycans. Individual sugar residues that may extend O-fucose, O-glucose or O-GlcNAc to varying degrees are: yellow circle, galactose; pink diamond, sialic acid; orange star, xylose. (B) Flow cytometry of Lec1 CHO cells expressing vector control or Eogt siRNA with NOTCH1 mAb, DLL1-Fc, DLL4-Fc or JAG1-Fc. (C) Relative mean fluorescence index (MFI) for binding of DLL1-Fc and DLL4-Fc to control and Eogt-siRNA Lec1 cells. Concentrations of ligand varied from 100 to 750 ng/ml. MFI values for binding to control cells taken as 1.0 were 455 ± 55 (DLL1-Fc) and 1215 ± 49 (DLL4-Fc). Data from 9 to 10 independent experiments are average, normalized MFI ± SEM; significance determined by paired, two-tailed Student’s t-test, *p<0.05, ****p<0.0001. (D) MFI values obtained for binding of DLL1-Fc or DLL4-Fc (750 ng/ml) to control and Eogt knockdown Lec1 CHO cells, before and after transfection of a human EOGT cDNA. Data are mean ± SEM from three independent experiments. Significance determined by unpaired, two-tailed Student’s t-test, *p<0.05. Western blot analysis of transfectants. (E) DLL4 and JAG1 beads bound to wild-type, EOGT-null, or NOTCH1-null HEK293T cells were observed by microscopy and counted (n = 50). Data are mean ± S.D. from three independent experiments. Statistical analysis was by Welch's t-test. **p<0.01; ***p<0.001. (F) Wild-type or EOGT-null HEK293T cells were transfected with Notch1 alone or together with Eogt followed by incubation with DLL4 beads. The number of DLL4 beads bound to cells was markedly increased by co-transfection of Eogt and Notch1. (G) Wild-type or EOGT-null HEK293T cells or cells transiently transfected with Notch1 with or without Eogt, were incubated with DLL4 or JAG1 beads. The number of Dynabeads bound per transfected cell marked by GFP expression was determined (n = 50). Data are mean ± S.D. from three independent experiments. *p<0.05; **p<0.01; ***p<0.001; #p<0.05; ###p<0.001 compared with the left-most wild type (*) or EOGTnull (#) bar by Welch's t test. (H) Wild-type or EOGT-null HEK293T cells were transfected with Notch1 with or without Eogt and subjected to flow cytometry using 8G10 NOTCH1 Ab. Mock transfectants were analyzed with (Mock) or without primary antibody (Cont).DOI: http://dx.doi.org/10.7554/eLife.24419.00310.7554/eLife.24419.004Figure 1—source data 1.Raw data for Figure 1C,D,E,G.DOI: http://dx.doi.org/10.7554/eLife.24419.004

Fig 3: O-GlcNAc on NOTCH1 EGF repeats promotes DLL4-NOTCH1 interactions.(A) Ala substitution of Thr/Ser in the O-GlcNAc consensus site C5XXG(Y/F)(T/S)GXXC6 in EGF2, 10, 17, and 20 in the NOTCH1?4xO-GlcNAc mutant. (B) Cell surface expression of NOTCH1?4xO-GlcNAc is comparable to that of wild-type NOTCH1. EOGT-null transfectants expressing Notch1 or Notch1?4xO-GlcNAc with or without Eogt were subjected to flow cytometry using 8G10 NOTCH1 antibody. Control transfectants were with (Mock) or without (Cont) primary antibody. Similar results were obtained for wild-type HEK293T cells (not shown). (C) Expression and O-GlcNAcylation of Notch1, Notch1?2xO-GlcNAc and Notch1?4xO-GlcNAc were analyzed by immunoprecipitation (IP) using NOTCH1 (8G10) antibody, followed by immunoblotting with CTD110.6 O-GlcNAc or NOTCH1 antibodies. (D) HEK293T transfectants expressing NOTCH1, NOTCH1?2xO-GlcNAc or NOTCH1?4xO-GlcNAc were immunostained for O-GlcNAc (CTD110.6 mAb; red) or NOTCH1 (8G10 mAb; green). Merged images include DAPI (blue) staining. Note that CTD110.6 mAb binding is markedly decreased in the NOTCH1?4xO-GlcNAc mutant. (E) Quantification of the number of DLL4- or JAG1-coated Dynabeads bound to Notch1 versus Notch1?4xO-GlcNAc transfected cells (marked with GFP), in the presence and absence of Eogt. Data are mean ± S.D from three independent experiments. Each experiment analyzed 50 cells. *p<0.05; **p<0.01; ***p<0.001 (Welch's t test).DOI: http://dx.doi.org/10.7554/eLife.24419.00610.7554/eLife.24419.007Figure 2—source data 1.Raw data for Figure 2E.DOI: http://dx.doi.org/10.7554/eLife.24419.007

Fig 4: EOGT acts on Notch receptors to regulate ligand-induced Notch signaling in ECs.(A) Relative mRNA expression in purified brain EC cells. WT (gray) and Eogt-/- (dark gray) EC cells isolated from cerebrum using anti-CD31 antibody were analyzed for gene expression related to the Notch signaling pathway. Gene transcript levels were normalized to Gapdh. Data are mean ± S.D. from three independent experiments performed in triplicate. Each experiment analyzed pooled total RNA obtained from 10 mice. *p<0.05; **p<0.01 (Welch's t test). (B) qRT-PCR analysis of Hes1 and Hey1 in wild type or Eogt-/- lung EC cells following stimulation with immobilized DLL4-Fc or JAG1-Fc. Gene transcripts were normalized to Gapdh. Data are mean ± S.D. from three independent experiments performed in triplicate. Each experiment analyzed total RNA obtained from a single mouse. Significance determined by Welch's t-test. *p<0.05; **p<0.01; #p<0.05; ##p<0.01 compared with the left-most Hes1 (*) or Hey1 (#) histogram. (C) Cells were transfected to express Dll4 or Jag1 and subjected to flow cytometry using DLL4 or JAG1 antibody, respectively. Comparison with mock-transfected cells indicated the amount of exogenously expressed DLL4 and JAG1 on the cell surface in wild-type or EOGT-null HEK293T cells. Mock-transfected HEK293T cells were labeled without primary antibody (Cont.). (D) Dll4/Jag1-transfected cells or Mock transfectants (signal-sending cells) were co-cultured with wild type or Eogt-/- lung EC cells (signal-receiving cells). qRT-PCR analysis of mouse Hey1 expression suggested that EOGT is required for the ability to receive DLL4-mediated Notch signaling, and that EOGT is dispensable for DLL4 and JAG1 as inducers of Notch signaling. Gene transcript levels were normalized to PECAM1 (CD31). Data are mean ± S.D. from three independent experiments performed in triplicate. Significance determined by Welch's t-test. ***p<0.001.DOI: http://dx.doi.org/10.7554/eLife.24419.02110.7554/eLife.24419.022Figure 8—source data 1.Raw data for Figure 8A,B,D.DOI: http://dx.doi.org/10.7554/eLife.24419.022

Fig 5: MenaINV expression in tumor cells induced by macrophages depends partially on TNFα-mediated NF-κB signaling and Notch1 Jagged1 signaling. A MenaINV mRNA expression in MDA-MB-231 (231) cells co-cultured with or without BAC2.1F macrophages (Mac) and with or without C87 (TNFα inhibitor) or SAHM1 (MAML1 inhibitor) for 4 h. B MenaINV mRNA expression in 231 cells co-cultured with or without Macs, TNFα inhibitor (C87), or Jag 1 or Jag2 blocking antibodies for 4 h. Bars in A and B represent average fold change of MenaINV mRNA expression compared to control cells (231). C MenaINV mRNA expression in 231 cells co-cultured with sgControl (WT) or Jagged1 knockout BAC1.2F5 macrophages (Jag1 KO Macs). Bars in (A–C) represent average fold change of MenaINV mRNA expression compared to control cells (A and B: 231; C: 231 + WT Macs). Data were analyzed using a one-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. = not significant. All experiments were repeated at least three times