Journal: Archives of biochemistry and biophysics

Article Title: UGDH promotes 5-fluorouracil resistance in colorectal cancer via the ROS-activated PI3K/AKT-EEF1A2-PRDX1 pathway.

doi: 10.1016/j.abb.2025.110445

Figure Lengend Snippet: Fig. 7. ROS activate the PI3K/AKT pathway, regulating the UGDH-EEF1A2-PRDX1 axis to promote chemoresistance. (A) KEGG pathway enrichment of DEGs in UGDH-knockdown SW620 cells (n = 3 replicates). (B) Western blot of PI3K/AKT pathway proteins in HCT8 cells ± LY294002 (10 μM, 48 h, n = 3). (C) PI3K/AKT phosphorylation in HCT8 control and shUGDH cells ± 5-FU (n = 3). (D) UGDH and PI3K/AKT activation in HCT8 cells treated with H2O2 (0–200 μM, 24 h, n = 3). (E) Time-dependent UGDH activation in HCT8 cells treated with 15 μM 5-FU (0–96 h). (n = 3). (F) Western blot of PI3K/AKT pathway proteins in HCT8 cells ± LY294002 (10 μM, n = 3)/± NAC (750 μМ, n = 3). Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The primary antibodies used were against UGDH (Proteintech), EEF1A2 (Proteintech), PRDX1 (Proteintech), PI3K (CST), phospho-PI3K (CST), Akt (CST), phospho-AKT (Ser473) (CST), FLAG (Proteintech), β-ACTIN (Proteintech), GAPDH (Beyotime), cleaved caspase 3 (CST), and cleaved PARP (CST).

Techniques: Knockdown, Western Blot, Phospho-proteomics, Control, Activation Assay

Journal: bioRxiv

Article Title: Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis

doi: 10.1101/2021.09.24.461701

Figure Lengend Snippet: A) Microarray heatmap shows P2RY14 receptor expression in p75 + /EGFR + SCP-like tumor initiating cells derived from human plexiform neurofibroma tumor cells compared to p75 + /EGFR - SCP-like cells. B) Western blot of human Schwann cells and neurofibroma SCP shows the latter has a 1.9-fold increase in P2RY14 protein expression. C) Representative FACS plot shows live sorted human plexiform neurofibroma tumor cells. D) Representative FACS plot shows human plexiform neurofibroma tumor cells sorted into p75 + /EGFR + SCP-like tumor initiating cells (pink square). E) Representative FACS plot shows p75 + /EGFR+ SCP-like tumor initiating cells further sorted into p75 + /EGFR + /P2RY14 - (left, purple square) and P75 + /EGFR + /P2RY14 + (right, blue square). F) Photomicrographs of human neurofibromas dissociated using FACS to yield: p75 + /EGFR + /P2RY14 - and P75 + /EGFR + /P2RY14 + cells. G) Quantification of unsorted, p75 + /EGFR + /P2RY14 - and P75 + /EGFR + /P2RY14 + cells plated in sphere medium. (n=3; two-way ANOVA; primary: **p=0.0057, ****p<0.0001; secondary: *p=0.0487,**p<0.0024, ****p<0.0001; tertiary:*p=0.0321, ***p=0.0006).

Article Snippet: For cell sorting, we incubated cell suspensions with anti-P2RY14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018) on ice for 30 minutes, washed with PBS twice.

Techniques: Microarray, Expressing, Derivative Assay, Western Blot

Journal: bioRxiv

Article Title: Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis

doi: 10.1101/2021.09.24.461701

Figure Lengend Snippet: A) Western blot shows Nf1 -/- SCPs have 1.5-fold change increase in P2RY14 expression. B) Quantification of percent of sphere forming cells in mouse WT and Nf1 -/- SCPs treated with the selective P2RY14 inhibitor (300nM PPTN) (primary, secondary, tertiary passage) (n=3; two-way ANOVA; primary: *p=0.0375, ***p=0.0001, ****p<0.0001; secondary: *p=0.0101, **p=0.0015, ***p=0.0009; tertiary: *p=0.0101, **p=0.0050, ***p=0.0005). C) P2RY14 mRNA expression in WT and Nf1 -/- E12.5 mouse SCP treated with shnon-target (shNT) control and shP2RY14 (****p<0.0001). D) Western blot of WT and Nf1 -/- SCPs treated with shNT and shP2RY14 showing P2RY14 knockdown. WT shP2RY14 show a 0.4-fold decrease of P2RY14 protein compared to WT shNT; and Nf1 -/- shP2RY14 show a 0.5-fold decrease compared to Nf1 -/- . E) Quantification of percent of sphere forming cells in mouse WT and Nf1 -/- SCPs treated with shNT and shP2RY14 (n=3; two-way ANOVA; primary: *p=0.0288, ****p<0.0001; secondary: **p=0.0029,***p=0.0005, ****p<0.0001; tertiary: *p=0.0154, ***p=0.0007). F) Western blot of WT and Nf1 -/- SC spheres shows 0.73-fold decrease of pERK in WT cells and 0.58-fold pERK decrease in Nf1 -/- cells after P2RY14 knockdown. G) Western blot of WT and Nf1 -/- SC spheres shows changes in pPKA substrate phosphorylation after shP2RY14 knockdown. WT shP2RY14 show a 1.43-fold increase in pPKA after P2RY14 knockdown; Nf1-/- cells have a 1.31-fold increase in pPKA expression after P2RY14 knockdown. H) Quantification of percent of sphere forming cells in Nf1 -/- mouse SCPs treated with 1uM rolipram or 300nM PPTN (n=3; two-way ANOVA; primary: ***p=0.0002, ****p<0.0001; secondary **p=0.0030, ****p<0.0001; tertiary: *p=0.0476, ***p=0.0004).

Article Snippet: For cell sorting, we incubated cell suspensions with anti-P2RY14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018) on ice for 30 minutes, washed with PBS twice.

Techniques: Western Blot, Expressing, Control, Knockdown, Phospho-proteomics

Journal: bioRxiv

Article Title: Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis

doi: 10.1101/2021.09.24.461701

Figure Lengend Snippet: A) Photomicrographs of WT and Nf1-/- mouse SC spheres treated with the selective P2RY14 inhibitor (PPTN; 300nM). B) Photomicrographs of WT and Nf1-/- mouse SC spheres treated with shNT and shP2RY14 (09). C) Photomicrographs of NF1-/- SCPs treated with vehicle and 1uM of rolipram.

Article Snippet: For cell sorting, we incubated cell suspensions with anti-P2RY14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018) on ice for 30 minutes, washed with PBS twice.

Techniques:

Journal: bioRxiv

Article Title: Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis

doi: 10.1101/2021.09.24.461701

Figure Lengend Snippet: A) Photomicrographs of WT and Nf1-/- mouse SC spheres treated with the P2RY14 inhibitor (PPTN; 30nM). B) Photomicrographs of WT and Nf1-/- mouse SC spheres treated with the P2RY14 inhibitor (PPTN; 100nM). C) Photomicrographs of WT and Nf1-/- mouse SC spheres treated with the P2RY14 inhibitor (PPTN; 500nM). (Note: for figures S2A-C, the same WT and Nf1-/- vehicle controls photomicrographs are used for each dose). D) Quantification of the percent of sphere forming cells after treatment with the P2RY14 inhibitor (PPTN; 30nM) in WT and Nf1-/- mouse SC spheres (two-way ANOVA: primary and secondary passage: ****p<0.0001, tertiary passage: *p=0.0168, **p=0.0074) E) Quantification of percent sphere forming cells after treatment with the P2RY14 inhibitor (PPTN;100nM) in WT and Nf1-/- mouse SCP (two way ANOVA: primary, secondary and tertiary passage: ****p<0.0001). F) Quantification of percent sphere forming cells after treatment with the P2RY14 inhibitor (PPTN; 500nM) in WT and Nf1-/- mouse SCP; results show that this concentration was toxic to the cells (two way ANOVA: primary, secondary and tertiary passage: ****p<0.0001).

Article Snippet: For cell sorting, we incubated cell suspensions with anti-P2RY14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018) on ice for 30 minutes, washed with PBS twice.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis

doi: 10.1101/2021.09.24.461701

Figure Lengend Snippet: A) Growth curve of iHSC1λ and iHSC1λ NF1#1 (1λ Δ NF). Top right inset shows western blot confirming NF1 protein expression in iHSC 1λ (positive control), ipNF95.11bc SCs (negative control) and iHSC 1λ NF1#1 (1λ Δ NF) (experimental). B) EdU incorporation assay in iHSC1λ and iHSC1λ NF1#1 (1λ Δ NF) (*p<0.001). C) 1λ and 1λ ΔNF1 treated with a potent 10uM UDP analog (P2RY14 specific agonist MRS2690), 500nM of the P2RY14 inhibitor (PPTN) and 200uM of IBMX (n=3). Western blot show changes in p-CREB expression upon treatment. D) 1λ and 1λΔNF1 cells treated with the P2RY14 inhibitor (PPTN; 500nM) at different timepoints (0,5,15,30,60,120 minutes) after EGF stimulation and western blot shows changes in pEGFR-Grb2 (Y1068), total EGFR, pERK1/2, total ERK1/2, p-AKT, total AKT and β-actin. E) Western blot shows pERK1/2, ERK1/2 and pAKT changes in ST88-14 cells treated with different concentrations of the P2RY14 inhibitor (PPTN) (0, 62.5nM, 125nM, 250nM and 500nM of PPTN) and 20ng/mL of EGF. F) Western blot shows ST88-14 cells treated with the P2RY14 inhibitor (PPTN; 500nM) and EGF (20ng/mL) and shows changes in EGFR phosphorylation at pEGFR-Grb2 (Y1068) binding site and changes in pERK and pAKT. G) ST88-14 cells transfected with P2RY14-GFP and/or EGFR-myc tagged proteins and immunoprecipitated EGFR using an anti-myc antibody shows the formation of a P2RY14 complex with EGFR.

Article Snippet: For cell sorting, we incubated cell suspensions with anti-P2RY14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018) on ice for 30 minutes, washed with PBS twice.

Techniques: Western Blot, Expressing, Positive Control, Negative Control, Phospho-proteomics, Binding Assay, Transfection, Immunoprecipitation

Journal: bioRxiv

Article Title: Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis

doi: 10.1101/2021.09.24.461701

Figure Lengend Snippet: A) Graph shows total EGFR and pEGFR (Grb2) dynamics overtime after addition of EGF in vehicle treated 1λ cells. B) Graph shows total EGFR and pEGFR (Grb2) dynamics overtime after addition of EGF in P2RY14 inhibitor (PPTN) treated 1λ cells. C) Graph shows total EGFR and pEGFR (Grb2) dynamics overtime after addition of EGF in vehicle treated 1λΔNF1 cells. D) Graph shows total EGFR and pEGFR (Grb2) dynamics overtime after addition of EGF in P2RY14 inhibitor (PPTN) treated 1λΔNF1 cells. E) Graph shows pERK1/2 and pAKT dynamics overtime after addition of EGF in vehicle treated 1λ cells. F) Graph shows pERK1/2 and pAKT dynamics overtime after addition of EGF in P2RY14 inhibitor (PPTN) treated 1λ cells. G) Graph shows pERK1/2 and pAKT dynamics overtime after addition of EGF in vehicle treated 1λΔNF1 cells. H) Graph shows pERK1/2 and pAKT dynamics overtime after addition of EGF in P2RY14 inhibitor (PPTN) treated 1λΔNF1 cells.

Article Snippet: For cell sorting, we incubated cell suspensions with anti-P2RY14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018) on ice for 30 minutes, washed with PBS twice.

Techniques:

Journal: bioRxiv

Article Title: Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis

doi: 10.1101/2021.09.24.461701

Figure Lengend Snippet: A) Schematic of generation of neurofibroma mice breeding of P2RY14 -/- mice with Nf fl/fl mice to obtain P2RY14 +/- ;Nf1fl/fl;Dhh+ and P2RY14 -/- ;Nf1fl/fl ; Dhh+ after several crosses. B) Genotyping confirmation of wildtype (WT) and P2RY14 knockout (KO) alleles. C) Western blot of sciatic nerve and neurofibroma tumors of Nf1fl/fl;Dhh+ and P2RY14 -/- ;Nf1fl/fl ; Dhh+ mice show decrease in P2RY14 expression upon P2RY14 knockdown (1 to 0.1-fold decrease in sciatic nerve and 1 to 0.2-fold decrease in neurofibroma tissue). D) Spinal cord (SC) immunofluorescent staining of mouse embryos at E12.5 shows P2RY14 expression (β-galactosidase) co-localization with SOX-10 SCs at dorsal and ventral roots (VR). E) Spinal cord (SC) immunofluorescent staining of mouse embryos at E12.5 shows P2RY14 expression (β-galactosidase) co-localization with SOX-10 SCs at dorsal root ganglion (DRG). E1 and E2 insets show zoomed picture of DRG. F) Immunofluorescent staining of 7-month-old mouse sciatic nerve shows β-galactosidase positive staining as a confirmation of P2RY14 knock-in; co-labeling of β-galactosidase and CNPase shows that P2RY14 co-localizes with SCs (inset). G) DAB staining of 7-month old WT nerve and Nf1fl/fl;Dhh+ mouse neurofibromas (DAB staining: brown (P2RY14 positive cells) blue (cell nuclei)).

Article Snippet: For cell sorting, we incubated cell suspensions with anti-P2RY14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018) on ice for 30 minutes, washed with PBS twice.

Techniques: Knock-Out, Western Blot, Expressing, Knockdown, Staining, Knock-In, Labeling

Journal: bioRxiv

Article Title: Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis

doi: 10.1101/2021.09.24.461701

Figure Lengend Snippet: A) Kaplan Meier survival plot of Nf1fl/fl;Dhh+ (red line; n=8); P2RY14 +/- ;Nf1fl/fl;Dhh+ (black line, n=14); P2RY14 -/- ;Nf1fl/fl ; Dhh+ (blue line; n=13); Nf1fl/+ control (green line, n=11) (*p=0.0256). B) Representative image of gross dissection of Nf1fl/fl ; Dhh+ and P2RY14 -/- ;Nf1fl/fl ; Dhh+ mice at 7-months of age. C) Neurofibroma tumor number quantification at 7-months of age (unpaired t test ****p<0.0001). D) Neurofibroma diameter quantification at 7-months (unpaired t test ***p=0.0004) (for figures C & D: Nf1fl/fl ; Dhh + n=8 mice, 48 neurofibroma tumors; P2RY14-/-;Nf1fl/fl ; Dhh+ n=8 mice, 11 neurofibroma tumors). E) Ki67 staining of mouse DRG and neurofibroma tissue at 7-months of age. F) Quantification of Ki67+ cells in mouse DRG and neurofibroma tissue at 7-months of age (One-way ANOVA; multiple comparisons ***p=0.0008; ****p<0.0001). G) Immunohistochemistry of human neurofibroma shows P2RY14 expression (DAB staining: brown (P2RY14 positive cells) blue (cell nuclei)). H) anti p-PKA substrate staining in WT , Nf1fl/fl;Dhh+ and P2RY14 -/- ;Nf1fl/fl,Dhh+ mice. p-PKA substrate phosphorylation labeling co-localized with CNPase SC marker (insets). I) Western blot of tissue lysates of WT (DRG), Nf1fl/fl;Dhh+ (neurofibroma tumors) and P2RY14 -/- ;Nf1fl/fl ; Dhh+ (neurofibroma tumors) show Nf1fl/fl;Dhh+ neurofibromas have 1.5-fold increased p-ERK expression compared to WT DRGs but, p-ERK expression between Nf1fl/fl;Dhh+ (1.5-fold) and P2RY14 -/- ;Nf1fl/fl ; Dhh+ (1.6-fold) remained relatively unchanged.

Article Snippet: For cell sorting, we incubated cell suspensions with anti-P2RY14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018) on ice for 30 minutes, washed with PBS twice.

Techniques: Control, Dissection, Staining, Immunohistochemistry, Expressing, Phospho-proteomics, Labeling, Marker, Western Blot

Journal: bioRxiv

Article Title: Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis

doi: 10.1101/2021.09.24.461701

Figure Lengend Snippet: Tumor dissection of Nf1fl/fl ; Dhh+ and P2RY14 -/- ;Nf1fl/fl ; Dhh+ mice at 4-months and immunostaining analysis of 4-months and 7-months sciatic nerve and tumors. A) Representative image of gross dissection of Nf1fl/fl ; Dhh+ and P2RY14 -/- ;Nf1fl/fl ; Dhh+ mice at 4-months. B) Neurofibroma tumor number quantification at 4-months of age (unpaired t-test **p=0.0052). C) Neurofibroma tumor diameter quantification at 4-months (unpaired t-test **p=0.0489) (for figures B & C: Nf1fl/fl ; Dhh+ n=5 mice, 19 neurofibroma tumors; P2RY14-/-;Nf1fl/fl ; Dhh+ n=5 mice; 5 neurofibroma tumors). D) Ki67 staining of Nf1fl/fl ; Dhh+ and P2RY14-/-;Nf1fl/fl ; Dhh+ neurofibromas at 4-months of age. E) Quantification of percent of Ki67 positive cells in Nf1fl/fl ; Dhh+ and P2RY14-/-;Nf1fl/fl ; Dhh+ 4-months old mice (t-test; n.s.). F) H&E staining at 4-months in Nf1fl/fl ; Dhh+ and P2RY14-/-;Nf1fl/fl ; Dhh+ mice. G) Ki67 immunofluorescence co-labeling with CNPase (SC marker) in 7-months sciatic nerve. H) H&E tumor staining at 7-months in Nf1fl/fl ; Dhh+ and P2RY14-/-;Nf1fl/fl ; Dhh+ mice.

Article Snippet: For cell sorting, we incubated cell suspensions with anti-P2RY14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018) on ice for 30 minutes, washed with PBS twice.

Techniques: Dissection, Immunostaining, Staining, Immunofluorescence, Labeling, Marker

Journal: bioRxiv

Article Title: Purinergic receptor P2RY14 cAMP signaling regulates EGFR-driven Schwann cell precursor self-renewal and nerve tumor initiation in neurofibromatosis

doi: 10.1101/2021.09.24.461701

Figure Lengend Snippet: A) Electron micrograph of saphenous nerve of 4-months WT, Nf1fl/fl ; Dhh, P2RY14 -/- ;Nf1fl/fl ; Dhh+ and P2RY14 -/- mice. B) Electron micrograph of saphenous nerve of 7-months WT, Nf1fl/fl ; Dhh and P2RY14 -/- ;Nf1fl/fl ; Dhh+ mice. C) Remak bundle quantification at 4-months of age (n=3; two way ANOVA: ****p<0.0001). D) Remak bundle quantification at 7-months of age (n=3; two way ANOVA: **p=0.0027, ****p<0.0001).

Article Snippet: For cell sorting, we incubated cell suspensions with anti-P2RY14 receptor antibody (Rabbit, polyclonal, Alomone labs, # APR-018) on ice for 30 minutes, washed with PBS twice.

Techniques:

Journal: Hormones & cancer

Article Title: Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential.

doi: 10.1007/s12672-016-0268-z

Figure Lengend Snippet: Fig. 2 Re-expression of AR-activated genes UGDH and PSA is dose dependent following prolonged androgen depletion. LNCaP 33 and 81 cells were selected for 10 days in the indicated DHTconcentrations. Prior to harvest, media were removed and replaced with media containing vehicle or 10 nM DHT for 24 h. a Equivalent amounts of protein were immunoblotted for UGDH and tubulin. UGDH expression was quantified, normalized to tubulin, and plotted as the ratio of 24 h DHT- treated versus vehicle-treated expression to illustrate stimulated expression. Mean ± SEM is plotted; *p < 0.05 relative to LNCaP 33 at 10 nM DHT; ‡p < 0.05 for LNCaP 81 relative to LNCaP 33 at the indicated DHT treatment. b Blots in (a) were re-probed for PSA (34 kDa) and AR (102 kDa)

Article Snippet: To generate UGDH knockdown lines, LNCaP 33 cells were transfected with plasmids encoding short hairpin RNA (shRNA) targeted to UGDH or a scrambled non-targeting control (UGDH shRNA-pGFP-V-RS no. TG334012 and 29-mer oligo-pGFP-V-RS no. TR30008, respectively, from Origene Technologies, Rockville, MD).

Techniques: Expressing

Journal: Hormones & cancer

Article Title: Loss of exogenous androgen dependence by prostate tumor cells is associated with elevated glucuronidation potential.

doi: 10.1007/s12672-016-0268-z

Figure Lengend Snippet: Fig. 7 UGDH knockdown reduces HSD3B1. LNCaP 33, 81, 22Rv1, and PC3 cell lines (a, c) or LNCaP 33 control and UGDH knockdown clones (b, d) were grown in standard steroid-replete media. Total cell lysates were normalized by protein content and analyzed in triplicate by immunoblots probed for HSD17B3 (a, b) or HSD3B1 (c, d). Densitometric quantification was normalized to tubulin and plotted as mean ± SEM of fold changes relative to LNCaP 33 or C1. *p < 0.05 relative to LNCaP 33; ‡p < 0.05 relative to C1

Article Snippet: To generate UGDH knockdown lines, LNCaP 33 cells were transfected with plasmids encoding short hairpin RNA (shRNA) targeted to UGDH or a scrambled non-targeting control (UGDH shRNA-pGFP-V-RS no. TG334012 and 29-mer oligo-pGFP-V-RS no. TR30008, respectively, from Origene Technologies, Rockville, MD).

Techniques: Knockdown, Control, Clone Assay, Western Blot

Journal: BMC Cancer

Article Title: Targeting INF2 with DiosMetin 7-O-β-D-Glucuronide: a new stratagem for colorectal cancer therapy

doi: 10.1186/s12885-025-14357-9

Figure Lengend Snippet: DiosMetin 7-O-β-D-Glucuronide had high affinity to INF2 protein: ( A ) The flow chat of computer virtual screening for INF2 protein. B Top5 FDA-approved drugs with highest docking score for INF2 protein. C Top5 natural products with highest docking score for INF2 protein. Docking model between Uridine 5′-diphosphoglucose ( D ), Alginic acid ( E ), DiosMetin 7-O-β-D-Glucuronide ( F ), 2-O-β-D-Glucopyranosyl-L-ascorbic acid ( G ), and GDP-D-mannose ( H ). I - J Cellular Thermal Shift Assay was performed to detect the interactions between molecules and INF2 protein. * represents p < 0.05; ** represents p < 0.01. n = 3. The DMSO treatment group was used for comparison. Data are shown as mean ± SD

Article Snippet: SW480 cells (2 × 10 5 cells) were subjected to treat with volume DMSO, 10 μM Uridine 5′-diphosphoglucose disodium salt (cat no. HY-N7032, MCE, Wuhan, China), 10 μM Alginic acid (cat no. HY-W127758, MCE), or 10 μM Diosmetin 7-O-β-D-Glucuronide (cat no. HY-N6879, MCE) in culture dishes for 24 h, followed by centrifugation at 6000 g for 2 min at 37 °C.

Techniques: Thermal Shift Assay, Comparison

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: The rabbit polyclonal antibody against UGDH (catalog number NBP1-90906, 1:200 dilution for immunofluorescence) was obtained from Novus.

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: The rabbit polyclonal antibody against UGDH (catalog number NBP1-90906, 1:200 dilution for immunofluorescence) was obtained from Novus.

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: The rabbit polyclonal antibody against UGDH (catalog number NBP1-90906, 1:200 dilution for immunofluorescence) was obtained from Novus.

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: The rabbit polyclonal antibody against UGDH (catalog number NBP1-90906, 1:200 dilution for immunofluorescence) was obtained from Novus.

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: The rabbit polyclonal antibody against UGDH (catalog number NBP1-90906, 1:200 dilution for immunofluorescence) was obtained from Novus.

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: The rabbit polyclonal antibody against UGDH (catalog number NBP1-90906, 1:200 dilution for immunofluorescence) was obtained from Novus.

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: The rabbit polyclonal antibody against UGDH (catalog number NBP1-90906, 1:200 dilution for immunofluorescence) was obtained from Novus.

Techniques: