Journal: Cell Death and Differentiation

Article Title: O-GlcNAcylation of UGDH regulates its activity and remodels the extracellular matrix to facilitate tumor growth

doi: 10.1038/s41418-025-01591-8

Figure Lengend Snippet: A TCGA analysis shows that UGDH expression is significantly upregulated in multiple cancer tissues, especially in hepatocellular carcinoma, breast invasive carcinoma, and NSCLC. B UGDH expression in NSCLC is higher in patients with stage III-IV than stage I-II according to the Barcelona criteria. C Kaplan–Meier analysis demonstrates that high levels of UGDH expression are associated with shorter survival outcomes in NSCLC. D UGDH has an evolutionarily conserved amino acid sequence across different species, which comprises residues of potential O-GlcNAcylation. E , F Analysis of UGDH glycosylation in HEK293T cells overexpressing Flag-tagged UGDH under co-transfection of HA-OGT or TMG treatment. G Immunoblotting analysis of the glycosylation ratio of UGDH using enzymatic labeling coupled with alkyne PEG-5kD. H , I The co-immunoprecipitation of OGT/OGA with UGDH. Immunoblotting analysis of UGDH glycosylation after different nutrient stimuli, including glucose ( J ), H 2 O 2 ( K ), glutamine ( L ) and EGF ( M ). N Immunoblotting analysis of UGDH expression and O-GlcNAcylation levels from primary NSCLC tissues and the matched adjacent tissues. Immunoblotting quantification of UGDH expression ( O ) and O-GlcNAcylation levels ( P ). Q Pearson’s correlation test was used to analyze the relationship between the levels of UGDH expression and UGDH O-GlcNAcylation. R Identification of O-GlcNAcylation sites on UGDH by high-resolution tandem mass spectrometry spectrum analysis. S Verifying the UGDH glycosylation sites using alanine mutations. Results are representative of three biological replicates.

Article Snippet: Specifically, the HRP-labeled monoclonal antibody against beta-actin (catalog number HRP-60008, 1:5000 dilution for immunoblotting) and the rabbit polyclonal antibody against UGDH (catalog number 13151-1-AP, 1:1000 dilution for immunoblotting) were purchased from Proteintech.

Techniques: Expressing, Sequencing, Glycoproteomics, Cotransfection, Western Blot, Labeling, Immunoprecipitation, Mass Spectrometry

Journal: Cell Death and Differentiation

Article Title: O-GlcNAcylation of UGDH regulates its activity and remodels the extracellular matrix to facilitate tumor growth

doi: 10.1038/s41418-025-01591-8

Figure Lengend Snippet: A Verification of UGDH deletion in the human A549 cell line. Two sgRNAs targeting UGDH fragments were amplified by PCR and cloned into the pMD™19-T vector for sequencing. Indels are marked in red. Three consecutive bases marked in red indicate the predicted Cas9 cleavage site. B A549 cells were depleted of endogenous UGDH by CRISPR-Cas9 and rescued with UGDH-WT or UGDH-S350A. C Representative images of the scratch assay at the indicated time performed in A549 cells rescued with UGDH-WT and UGDH-S350A are shown. D The transwell assay was used to quantitatively detect the migration ability of genetically modified A549 cells mentioned above. E – G A549 cells rescued with UGDH-WT or UGDH-S350A were implanted into C57BL/6Jnu/nu mice by tail vein injection to detect cancer cell metastasis ability in vivo. Representative images of H&E-stained sections in dissected lungs 21 days after injection are shown ( E ). The metastatic lesions are quantified based on the staining results of lung sections, the mean ± SEM of metastatic nodules in each of the 5 mice in each group ( F ). The overall survival (OS) of C57Bl/6Jnu/nu mice bearing lung metastases seeded with A549 cells with or without UGDH. n = 5 mice per group ( G ). H Schematic diagram of subcutaneous injection of LLC UGDH-WT and LLC UGDH-S350A cells and detection of primary tumor growth after 19 days. I Tumor growth curves of resultant UGDH-WT and UGDH-350A NSCLC tumors in C57Bl/6Jnu/nu or C57BL/6J mice. n = 5 mice per group. Statistics by student’s t -test. Tumor volumes were measured three times per week and calculated using the formula V = π/4 × length × width 2 . J Overall survival (OS) of C57Bl/6Jnu/nu or C57BL/6J mice bearing a primary tumor seeded with UGDH-WT or UGDH-350A cells. n = 5 mice per group. Statistics by Kaplan–Meier test. K Tumor graphs and weights are shown for NSCLC UGDH-WT and for UGDH-S350A growing in C57BL/6Jnu/nu or C57BL/6J mice at day 19 after s.c. injection. N = 5 per group. Data are presented as means ± SEM. Statistics by unpaired t -test.

Article Snippet: Specifically, the HRP-labeled monoclonal antibody against beta-actin (catalog number HRP-60008, 1:5000 dilution for immunoblotting) and the rabbit polyclonal antibody against UGDH (catalog number 13151-1-AP, 1:1000 dilution for immunoblotting) were purchased from Proteintech.

Techniques: Amplification, Clone Assay, Plasmid Preparation, Sequencing, CRISPR, Wound Healing Assay, Transwell Assay, Migration, Genetically Modified, Injection, In Vivo, Staining

Journal: Cell Death and Differentiation

Article Title: O-GlcNAcylation of UGDH regulates its activity and remodels the extracellular matrix to facilitate tumor growth

doi: 10.1038/s41418-025-01591-8

Figure Lengend Snippet: A A flow chart illustrates tissue processing and CyTOF analysis of lung tumors generated by subcutaneous injection. Two-dimensional T-distributed stochastic neighbor embedding (t-SNE) scatterplot of the CyTOF-TILs data derived from UGDH-WT ( B ) and UGDH-S350A ( C ) tumor showed that 8 main clusters were identified with corresponding markers. D Comparing the contributions of various clusters of immune cells from total CD45 + lymphocytes between the two groups of samples. E Representative immunohistochemistry images and quantification of CD8 + T cells in primary tumors formed by UGDH-WT and UGDH-S350A NSCLC cells. Scale bar, 100 μm. F t-SNE re-analysis of CD8 + T cells colored by relative expression of CyTOF markers, with clusters indicated. G Comparing the percentage of Granzyme B + CD8 + T cells in lymphocytes between UGDH-WT and UGDH-S350A tumors.

Article Snippet: Specifically, the HRP-labeled monoclonal antibody against beta-actin (catalog number HRP-60008, 1:5000 dilution for immunoblotting) and the rabbit polyclonal antibody against UGDH (catalog number 13151-1-AP, 1:1000 dilution for immunoblotting) were purchased from Proteintech.

Techniques: Generated, Injection, Derivative Assay, Immunohistochemistry, Expressing

Journal: Cell Death and Differentiation

Article Title: O-GlcNAcylation of UGDH regulates its activity and remodels the extracellular matrix to facilitate tumor growth

doi: 10.1038/s41418-025-01591-8

Figure Lengend Snippet: A Immunoblotting analysis of endogenous OGT depletion using shRNAs in A549 and NCI-H1299 cells. B ELISA analysis of HA concentrations in genetically engineered cells. C Immunofluorescence staining of HA accumulation from A549 cells upon UGDH depletion. HABP (HA stain in green) and DAPI (nuclear stain in blue). Scale bar, 10 μm. D Competitive ELISA analysis of the absolute concentration of HA in A549 cells with or without UGDH depletion. E Pathological staining and quantitative statistical analysis of the ECM contents. Masson’s trichrome staining for collagen or IHC staining for HA and α-SMA. The statistical data are shown as mean ± SEM ( n = 12–16). Statistical analyses were performed by an unpaired two-tailed Student’s t -test. F IHC analysis was performed with anti-UGDH, anti-HA, anti-collagen, anti-α-SMA, and anti-CD8 antibodies on specimens from patients with lung cancer. Representative images of IHC staining graphs from two patients are shown. Scale bar, 200 μm. G A higher level of UGDH O-GlcNAcylation, α-SMA, collagen, HA, and a lesser infiltration of CD8 + T cells in tumor tissues were observed compared to adjacent peritumoral tissues in a cohort of 18 patient samples. H The deposition of HA, α-SMA, and collagen increased with the level of UGDH O-GlcNAcylation, but not for CD8 + T cells. I The correlation between CD8 + T cell infiltration and the expression of HA. J The effect of CD8α mAb treatment on tumor growth. K Representative IHC images and quantification of CD8 + T cells in the tumors after CD8α mAb treatment. Scale bar, 100 μm. L 4-MU treatment diminished HA accumulation and abolished the effect of UGDH O-GlcNAcylation on tumor development.

Article Snippet: Specifically, the HRP-labeled monoclonal antibody against beta-actin (catalog number HRP-60008, 1:5000 dilution for immunoblotting) and the rabbit polyclonal antibody against UGDH (catalog number 13151-1-AP, 1:1000 dilution for immunoblotting) were purchased from Proteintech.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Competitive ELISA, Concentration Assay, Immunohistochemistry, Two Tailed Test, Expressing

Journal: Cell Death and Differentiation

Article Title: O-GlcNAcylation of UGDH regulates its activity and remodels the extracellular matrix to facilitate tumor growth

doi: 10.1038/s41418-025-01591-8

Figure Lengend Snippet: A In vitro establishment of UGDH enzymatic reaction ( n = 3). PBS group as a blank control, and the enzyme dead mutant UGDH T325D as a negative control. B Enzymatic activity of UGDH in the presence of OGT co-expression or TMG treatment (50 μM) in HEK293T cells ( n = 4). C Comparison of the enzyme activity of UGDH WT and UGDH S350A isolated from HEK293T cells in the presence of 50 μM TMG ( n = 3). D Comparison of the enzyme activity of UGDH WT and UGDH S350A isolated from Escherichia coli ( n = 4). E Steady-state kinetics of UGDH WT and UGDH S350A isolated from HEK293T for UDP-glucose ( n = 3). F The free energy profile as a function of the distance of UDP-glucose (UDP-Glc) from the binding sites, as obtained from the ten one-microsecond simulations. G The representative structure model of O-GlcNAcylated UGDH. The S350-O-GlcNAc and UDP-glucose are depicted in sticks, while their molecular surfaces are illustrated by dots in blue and magenta, respectively. NADH is represented in green sticks, and UGDH is shown in white cartoon form. Data are presented as means ± SEM. Statistical analyses were performed by an unpaired two-tailed Student’s t -test.

Article Snippet: Specifically, the HRP-labeled monoclonal antibody against beta-actin (catalog number HRP-60008, 1:5000 dilution for immunoblotting) and the rabbit polyclonal antibody against UGDH (catalog number 13151-1-AP, 1:1000 dilution for immunoblotting) were purchased from Proteintech.

Techniques: In Vitro, Control, Mutagenesis, Negative Control, Activity Assay, Expressing, Comparison, Isolation, Binding Assay, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: O-GlcNAcylation of UGDH regulates its activity and remodels the extracellular matrix to facilitate tumor growth

doi: 10.1038/s41418-025-01591-8

Figure Lengend Snippet: A Nuclear and cytoplasmic expression of STAT1 and p-STAT1 in UGDH WT and UGDH S350A LLC cells with or without IFNγ treatment. B , C STAT1 nuclear localization and quantification in rWT and rS350A LLC cells with or without IFNγ treatment ( n = 3). D Immunoblotting analysis of the association of HA-tagged UGDH with Flag-tagged STAT1. Immunoblotting analysis of the association of Flag-tagged KPNA2 ( E ) and His-tagged KPNB1 ( F ) with HA-tagged UGDH in HEK293T cells. G , H Immunoblotting analysis of the association of KPNA2 with UGDH WT and UGDH S350A in HEK293T cells in the presence of TMG or OSMI-4. I Immunoblotting analysis of the association of Flag-tagged KPNA2 with the endogenous STAT1 in HEK293T cells in the presence of UGDH WT or UGDH S350A. J Immunoblotting analysis of the association of Myc-tagged KPNA2 and Flag-tagged STAT1 in HEK293T cells in the presence of HA-UGDH WT or HA-UGDH S350A. K Nuclear and cytoplasmic STAT1 and p-STAT1 expressions in UGDH WT or UGDH-KO cells after IFNγ treatment. L Immunoblotting analysis of the association of endogenous STAT1 and KPNA2 in UGDH WT and KO cells with IFNγ treatment. Data are presented as means ± SEM. Statistical analyses were performed by an unpaired two-tailed Student’s t -test.

Article Snippet: Specifically, the HRP-labeled monoclonal antibody against beta-actin (catalog number HRP-60008, 1:5000 dilution for immunoblotting) and the rabbit polyclonal antibody against UGDH (catalog number 13151-1-AP, 1:1000 dilution for immunoblotting) were purchased from Proteintech.

Techniques: Expressing, Western Blot, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: O-GlcNAcylation of UGDH regulates its activity and remodels the extracellular matrix to facilitate tumor growth

doi: 10.1038/s41418-025-01591-8

Figure Lengend Snippet: Schematic model of how O-GlcNAcylation of UGDH contributes to immune regulation and tumor development.

Article Snippet: Specifically, the HRP-labeled monoclonal antibody against beta-actin (catalog number HRP-60008, 1:5000 dilution for immunoblotting) and the rabbit polyclonal antibody against UGDH (catalog number 13151-1-AP, 1:1000 dilution for immunoblotting) were purchased from Proteintech.

Techniques: