ogt inhibitor-st045849  (TimTec LLC)

Journal: bioRxiv

Article Title: O-GlcNAcylation and low glycolysis underpin Th2 polarization by dendritic cells

doi: 10.64898/2026.02.03.703465

Figure Lengend Snippet: a, Schematic of the hexosamine biosynthetic pathway (HBP) and O-GlcNAcylation. b, Relative mRNA expression of 24h-conditioned human moDCs based on bulk RNA sequencing data . c , Relative intracellular metabolite abundance of 24h-conditioned human moDCs based on flow injection analysis mass spectrometry (FIA-MS; ). d, Volcano plot showing differences in intracelullar metabolite abundance between Th2-DCs and iDCs. Positive values corresponding to metabolites with increased abundance in Th2-DCs. Significantly different metabolites are highlighted in red. e , Integration of metabolomic and transcriptomic data sets using CoMBI-T profiling related to the HBP pathway (as described in ). Round nodes represent metabolites within the core regulatory network. Enzymes are represented by edges. Differential expression of enzymes or abundance of metabolites are indicated by red (higher in Th2-DCs) to green (higher in iDCs) color scale. f, Flow cytometry-based analysis of overall protein O-GlcNAcylation of conditioned human moDCs. g, i-k, Human moDCs were preincubated with inhibitors of O-GlcNAcylation (ST045849 – OGTi), or N-glycosylation (tunicamycin) for 30 minutes, before stimulation with g , omega-1, i , House dust mite (HDM) extract, 2-DG, j , LPS/Poly-IC or Zymosan, after which washed DCs were cocultured with ( g-j ) allogeneic naïve Th cells and 11 days later Th1/2 cell polarization was determined by intracellular cytokine staining or k , with memory Th cells and 5 days after DC-T cell coculture and IL-17 concentrations were determined by ELISA. ( G ) Representative flow plots are shown on the left. On the right % cytokine-producing T cells are shown relative to control conditioned-DCs. h, Differentiating moDCs were transduced with short interfering RNA against O-GlcNAc transferase (OGT) at day 4 and subsequently analyzed as in ( g ). Scrambled RNA was used as a control. Data points represent independent experiments of individual donors. Bars represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001. Statistical significance was determined using one-way ANOVA with Dunnet post-hoc test ( f,g,i-k ) and Student’s t -test ( h ).

Article Snippet: Inhibitors included: 5-10 mM of 2-deoxyglucose (2-DG; in mQ water; #D8375-1g, Sigma), 20 μM ST045849 (ST; in DMSO; #6775, Tocris) and 10 μM Thiamet G (TG; in DMSO; #13237, Cayman Chemical, Ann Arbor, Michigan, United States).

Techniques: Expressing, RNA Sequencing, Injection, Mass Spectrometry, Metabolomic, Quantitative Proteomics, Flow Cytometry, Glycoproteomics, Staining, Enzyme-linked Immunosorbent Assay, Control, Transduction, Small Interfering RNA

Journal: bioRxiv

Article Title: O-GlcNAcylation and low glycolysis underpin Th2 polarization by dendritic cells

doi: 10.64898/2026.02.03.703465

Figure Lengend Snippet: ( A ) MS/MS based proteomic analysis of O-GlcNAcylated proteins in differently conditioned human moDCs. Volcano plot showing differential protein O-GlcNAcylation in Th2-DCs compared with iDCs. ( B ) Protein–protein interaction network of proteins exhibiting increased or decreased O-GlcNAcylation in Th2-DCs. ( C ) Gene Ontology (GO) enrichment analysis of cellular component terms associated with differentially O-GlcNAcylated proteins in Th2-DCs. ( D ) Confocal microscopy of F-Actin (green), Zyxin (red), and merged images of moDCs left untreated or stimulated for 24 h with LPS, omega1, or omega1 preincubated with the O-GlcNAcylation inhibitor ST045849 (OGTi) for 30 minutes. ( E ) quantification of the percentage of overlap between F-Actin and ZYXIN in individual cells. ( F ) Conditioned human moDCs were co-cultured with allogenic naïve Th cells for 4h. DC-T cell conjugates were identified as CD1a + CD4 + dual-positive events and quantified as the percentage of total DCs. ( G ) A schematic diagram for Atomic Force Microscopy (AFM)-based single-cell force spectroscopy (SCFS) assay setup. ( H ) DC2.4 cells were stimulated with LPS or omega1 with or without pre-incubated with OGTi prior to stimulation. Adhesion was quantified as in (G). ( I ) WT, Fascin ko or Zxyin ko DC2.4 cells were stimulated with LPS or omega1 with or without pre-incubated with OGTi prior to stimulation. Adhesion assay following knockout as in (G) ( J ) siRNA mediated knockdown of Fascin-1 or Zyxin was performed on day 4 of moDCs differentiation; scrambled siRNA served as control. On day 6, DCs were co-cultured with naïve allogeneic T cells for 11 days. Intracellular IFN-γ and IL-4 production was assessed by flow cytometry after 6h of PMA/ionomycin stimulation. Percentages of Th cells expressing IFN-γ or IL-4 are shown relative to untreated scrambled control. ( A-C ) Analysis based on data from 4 donors. One representative of ( D ) 4 or ( H,I ) 2 independent experiments is shown. ( F,J ) Datapoints represent individual donors from independent experiments. Data are shown as mean ± SEM. Statistical significance was determined by (E) paired Student’s t-test or (G-I) using two-way ANOVA with Tukey post-hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Inhibitors included: 5-10 mM of 2-deoxyglucose (2-DG; in mQ water; #D8375-1g, Sigma), 20 μM ST045849 (ST; in DMSO; #6775, Tocris) and 10 μM Thiamet G (TG; in DMSO; #13237, Cayman Chemical, Ann Arbor, Michigan, United States).

Techniques: Tandem Mass Spectroscopy, Confocal Microscopy, Cell Culture, Microscopy, Single Cell, Force Spectroscopy, Incubation, Cell Adhesion Assay, Knock-Out, Knockdown, Control, Flow Cytometry, Expressing