Journal: Oncogenesis

Article Title: O-GlcNAcylation of FBP1 promotes pancreatic cancer progression by facilitating its Lys48-linked polyubiquitination in hypoxic environments.

doi: 10.1038/s41389-025-00555-4

Figure Lengend Snippet: Fig. 1 Downregulation of FBP1 expression predicts a poor prognosis in patients with pancreatic cancer, and O-GlcNAcylation regulates FBP1 expression under hypoxia. A Representative IHC staining of 38 pairs of pancreatic tumour and normal adjacent tissues. Scale bar as shown. B Statistical analysis of FBP1 expression from the IHC results. We defined high expression as two points and low expression as 1 point. C Kaplan–Meier overall survival curves for all 38 patients with pancreatic cancer stratified by high and low FBP1 expression. D Correlations of FBP1 expression with T stage, lymph node metastasis, distant metastasis and AJCC TNM stage in clinical patients with pancreatic cancer according to the χ2 test. E Screening of FBP1 expression in pancreatic cancer cell lines. F Downregulation of FBP1 accelerates tumour growth in AsPC-1 and SW1990 cells. G FBP1 is repressed under hypoxia in AsPC-1 and SW1990 cells. H A hypoxic environment triggered the degradation of FBP1 in AsPC-1 and SW1990 cells.

Article Snippet: Liquid chromatograph-mass spectroscopy/mass spectroscopy (LC–MS/MS) The gels (the mixture pulled down by anti-Flag immunomagnetic beads) containing O-GlcNAcylated FBP1 or ubiquitinated FBP1 proteins were cut and then sent for LC–MS/MS to identify the modification sites at Shanghai Applied Protein Technology [26].

Techniques: Expressing, Immunohistochemistry

Journal: Oncogenesis

Article Title: O-GlcNAcylation of FBP1 promotes pancreatic cancer progression by facilitating its Lys48-linked polyubiquitination in hypoxic environments.

doi: 10.1038/s41389-025-00555-4

Figure Lengend Snippet: Fig. 2 OGT interacts with and O-GlcNAcylates FBP1 at the serine 47 residue. A Time course of FBP1 expression and hypoxia exposure time. B Treatment with siOGT or OSMI-1 reversed the degradation of FBP1 in AsPC-1 and SW1990 cells under hypoxia for 6 h. C A co-IP assay was performed to validate the interaction between exogenous FBP1/FBP1 (K908A) and OGT plasmids in 293 T cells. D A co-IP assay was performed to validate the interaction between endogenous FBP1 and OGT in AsPC-1 and SW1990 cells. E A PLA was performed to validate the interaction between endogenous FBP1 and OGT in AsPC-1 cells. The PLA signal was detected by fluorescence microscopy (red arrows). Scale bar as shown. F Four FBP1 truncations were constructed according to their structural domains. A co-IP assay was performed to determine the interacting domain. G Mass spectrometry results of the FBP1 serine 47 residue. H A co-IP assay between O-GlcNAc and the FBP1-WT/mutant was performed to validate the potential O-GlcNAcylation site. Endogenous FBP1 was knocked down and then rescued by Flag-tagged FBP1- WT, S47A, S67A, and S233A plasmids in AsPC-1 and SW1990 cells.

Article Snippet: Liquid chromatograph-mass spectroscopy/mass spectroscopy (LC–MS/MS) The gels (the mixture pulled down by anti-Flag immunomagnetic beads) containing O-GlcNAcylated FBP1 or ubiquitinated FBP1 proteins were cut and then sent for LC–MS/MS to identify the modification sites at Shanghai Applied Protein Technology [26].

Techniques: Residue, Expressing, Co-Immunoprecipitation Assay, Microscopy, Construct, Mass Spectrometry, Mutagenesis

Journal: Oncogenesis

Article Title: O-GlcNAcylation of FBP1 promotes pancreatic cancer progression by facilitating its Lys48-linked polyubiquitination in hypoxic environments.

doi: 10.1038/s41389-025-00555-4

Figure Lengend Snippet: Fig. 3 FBP1 O-GlcNAcylation at the serine 47 residue promotes its degradation in a hypoxic environment. A Silencing OGT with siRNAs regulates FBP1-WT/S47A protein expression levels in AsPC-1 and SW1990 cells. B Silencing OGT with siRNAs regulates FBP1-WT/S47A mRNA expression levels in AsPC-1 and SW1990 cells. C CHX assays were performed to determine the stability of the FBP1-WT/S47A proteins in OGT- knockdown AsPC-1 and SW1990 cells. D Hyper-O-GlcNAcylation by OGT or an OGT mutant (K908A) regulates FBP1-WT/S47A protein expression levels in AsPC-1 and SW1990 cells. E Hyper-O-GlcNAcylation by OGT or an OGT mutant (K908A) regulates FBP1-WT/S47A mRNA expression levels in AsPC-1 and SW1990 cells. F CHX assays were performed to determine the stability of the FBP1-WT/S47A proteins under hyper-O-GlcNAcylation by OGT in AsPC-1 and SW1990 cells. G Changes in FBP1-WT/S47A protein expression levels in AsPC-1 and SW1990 cells under hypoxic conditions. H CHX assays were performed to determine the stability of the FBP1-WT/S47A proteins in a hypoxic environment in AsPC-1 and SW1990 cells.

Article Snippet: Liquid chromatograph-mass spectroscopy/mass spectroscopy (LC–MS/MS) The gels (the mixture pulled down by anti-Flag immunomagnetic beads) containing O-GlcNAcylated FBP1 or ubiquitinated FBP1 proteins were cut and then sent for LC–MS/MS to identify the modification sites at Shanghai Applied Protein Technology [26].

Techniques: Residue, Expressing, Knockdown, Mutagenesis

Journal: Oncogenesis

Article Title: O-GlcNAcylation of FBP1 promotes pancreatic cancer progression by facilitating its Lys48-linked polyubiquitination in hypoxic environments.

doi: 10.1038/s41389-025-00555-4

Figure Lengend Snippet: Fig. 4 FBP1 O-GlcNAcylation at the serine 47 residue regulates its downstream target genes and promotes tumour progression. A RT‒ qPCR results showing the expression levels of downstream target genes in AsPC-1 and SW1990 (FBP1-WT/S47A) cells with or without OGT knockdown. B ChIP‒qPCR results showing FBP1 occupancy at the promoters of downstream target genes in AsPC-1 and SW1990 (FBP1-WT/ S47A) cells with or without OGT knockdown. C Glucose uptake assays were performed in AsPC-1 and SW1990 (FBP1-WT/S47A) cells with or without OGT knockdown. D Lactate secretion assays were performed in AsPC-1 and SW1990 (FBP1-WT/S47A) cells with or without OGT knockdown. E MTT assays were performed to measure the growth of AsPC-1 and SW1990 (FBP1-WT/S47A) cells with or without OGT knockdown. F Colony formation assays were performed in AsPC-1 and SW1990 (FBP1-WT/S47A) cells with or without OGT knockdown, and the statistical results were calculated. G Xenograft experiments in nude mice were performed in AsPC-1 and SW1990 (FBP1-WT/S47A) cells with or without OGT knockdown, and the statistical results (i.e., tumour weight and volume) were analysed.

Article Snippet: Liquid chromatograph-mass spectroscopy/mass spectroscopy (LC–MS/MS) The gels (the mixture pulled down by anti-Flag immunomagnetic beads) containing O-GlcNAcylated FBP1 or ubiquitinated FBP1 proteins were cut and then sent for LC–MS/MS to identify the modification sites at Shanghai Applied Protein Technology [26].

Techniques: Residue, Expressing, Knockdown

Journal: Oncogenesis

Article Title: O-GlcNAcylation of FBP1 promotes pancreatic cancer progression by facilitating its Lys48-linked polyubiquitination in hypoxic environments.

doi: 10.1038/s41389-025-00555-4

Figure Lengend Snippet: Fig. 5 FBP1 O-GlcNAcylation at the serine 47 residue promotes its TRIM28-mediated K48 polyubiquitination. A Cotransfection of OGT and an OGT mutant (K908A) revealed that OGT facilitates TRIM28-mediated FBP1 polyubiquitination in AsPC-1 and SW1990 wild-type cells. B Hypoxia facilitates TRIM28-mediated FBP1 polyubiquitination in AsPC-1 and SW1990 wild-type cells. C OGT facilitates TRIM28- mediated FBP1 K48-linked polyubiquitination in AsPC-1 and SW1990 wild-type cells. D OGT fails to affect TRIM28-mediated FBP1 K63- linked polyubiquitination in AsPC-1 and SW1990 wild-type cells. E Silencing TRIM28 abolishes OGT-mediated protein degradation in AsPC-1 and SW1990 wild-type cells. F Silencing TRIM28 abolishes hypoxia-mediated protein degradation in AsPC-1 and SW1990 wild- type cells.

Article Snippet: Liquid chromatograph-mass spectroscopy/mass spectroscopy (LC–MS/MS) The gels (the mixture pulled down by anti-Flag immunomagnetic beads) containing O-GlcNAcylated FBP1 or ubiquitinated FBP1 proteins were cut and then sent for LC–MS/MS to identify the modification sites at Shanghai Applied Protein Technology [26].

Techniques: Residue, Cotransfection, Mutagenesis

Journal: Oncogenesis

Article Title: O-GlcNAcylation of FBP1 promotes pancreatic cancer progression by facilitating its Lys48-linked polyubiquitination in hypoxic environments.

doi: 10.1038/s41389-025-00555-4

Figure Lengend Snippet: Fig. 6 FBP1 O-GlcNAcylation at the serine 47 residue promotes its K51 polyubiquitination. A Mass spectrometry results of the FBP1 serine 47 residue. B Co-IP between Ub and the FBP1-WT/lysine mutants was performed to validate the potential polyubiquitination site. C Conserved domains in the FBP1 protein in many species. D CHX assays were performed to determine the stability of the FBP1-WT/K51R proteins in AsPC- 1 and SW1990 cells. E Cotransfection of OGT and Ub plasmids revealed that blocking FBP1 O-GlcNAcylation at the serine 47 residue can reverse its K51 polyubiquitination in AsPC-1 and SW1990 cells. F Blocking the O-GlcNAcylation of FBP1 at the serine 47 residue can reverse the K51 polyubiquitination of FBP1 in AsPC-1 and SW1990 cells induced by a hypoxic environment.

Article Snippet: Liquid chromatograph-mass spectroscopy/mass spectroscopy (LC–MS/MS) The gels (the mixture pulled down by anti-Flag immunomagnetic beads) containing O-GlcNAcylated FBP1 or ubiquitinated FBP1 proteins were cut and then sent for LC–MS/MS to identify the modification sites at Shanghai Applied Protein Technology [26].

Techniques: Residue, Mass Spectrometry, Co-Immunoprecipitation Assay, Cotransfection, Blocking Assay

Journal: Oncogenesis

Article Title: O-GlcNAcylation of FBP1 promotes pancreatic cancer progression by facilitating its Lys48-linked polyubiquitination in hypoxic environments.

doi: 10.1038/s41389-025-00555-4

Figure Lengend Snippet: Fig. 7 FBP1 K51 polyubiquitination regulates its downstream target genes, and the O-GlcNAcylation/polyubiquitination axis promotes tumour progression. A RT‒qPCR results showing the expression levels of downstream target genes in AsPC-1 and SW1990 (FBP1-WT/K51R) cells with or without OGT knockdown. B Glucose uptake assays were performed in AsPC-1 and SW1990 (FBP1-WT/K51R) cells with or without OGT knockdown. C Lactate secretion assays were performed in AsPC-1 and SW1990 (FBP1-WT/K51R) cells with or without OGT knockdown. D MTT assays were performed to measure the growth of AsPC-1 and SW1990 (FBP1-WT/K51R) cells with or without OGT knockdown. E Colony formation assays were performed in AsPC-1 and SW1990 (FBP1-WT/K51R) cells with or without OGT knockdown, and the statistical results were calculated. F Xenograft experiments in nude mice were performed in AsPC-1 and SW1990 (FBP1-WT/K51R) cells with or without OGT knockdown, and the statistical results (i.e. tumour weight and volume) were analysed. G Schematic model of how the FBP1 O-GlcNAcylation/ polyubiquitination axis functions in pancreatic cancer cells in a hypoxic environment.

Article Snippet: Liquid chromatograph-mass spectroscopy/mass spectroscopy (LC–MS/MS) The gels (the mixture pulled down by anti-Flag immunomagnetic beads) containing O-GlcNAcylated FBP1 or ubiquitinated FBP1 proteins were cut and then sent for LC–MS/MS to identify the modification sites at Shanghai Applied Protein Technology [26].

Techniques: Expressing, Knockdown

Journal: BMC Cancer

Article Title: Fuse binding protein antagonizes the transcription activity of tumor suppressor protein p53

doi: 10.1186/1471-2407-14-925

Figure Lengend Snippet: FBP negatively affects p21 and BCCIP expression in Huh7 cells. (A) Stably FBP-knockdown (FBP-kd) Huh7 cells by expressing FBP1 targeting shRNA: Huh7 cells were transfected with lentivirus vector encoding FBP1 targeting shRNAs or with empty vector alone. Stable clones (NP-4, NP-5) were selected after several passages via puromycin selection and were confirmed for stable knockdown of FBP1 expression by Western blot analysis as compared to cells transformed with the vector alone (NP-2, NP-3). (B) . Expression of p53 and p21 in FBP-kd cells. Huh7 cells stably transduced with FBP1 shRNA (NP-4) or empty vector alone (NP-2) were grown for 48 h. Cell lysates were normalized with respect to protein and Western blotted for the expression of FBP1, p53, p21, and actin. Lane 1, control Huh7 cells; lane 2, FBP-kd Huh7 cells; lane 3, Huh7 cells transduced with a lentivirus vector only. (C) Construction of FBP1 expression clone resistant to FBP1-shRNA. The FBP1-shRNA targeted sequences spanning codons 248 to 254 and from 560 to 567 were subjected to point mutation in FBP1 expression clone (pCIA-CMV-FBP) without changing the amino acid sequence. (D) Transient expression of FBP1 in FBP-kd cells suppressed the expression of p21. FBP-kd cells were transfected with shRNA resistant FBP1 expression clone ((pCIA-CMV-FBP SHR ) or empty pCIA-CMV vector; 48-h later cells lysates were Western blotted for FBP1, p53 and p21 and Actin. Lane 1, control Huh7 cells, lane 2, Huh7 cells transduced with lentiviral vector alone; lane 3, FBP-kd cells; lane 4, FBP-kd cells transfected with empty vector; lane 5, FBP-kd cells transfected with shRNA resistant FBP1 expression clone. (E) Expression level of p21 in control and p53-kd, BCCIP-kd, and FBP-kd Huh7 cells. Lane1, control Huh7 cells; lane 2, lentivirus vector control; lane 3, p53-kd; lane 4, BCCIP-kd and lane 5, FBP-kd Huh7 cells. A colon cancer cell line, HCT116 without p53 (-/-) (lane 6) or with wild-type p53 (+/+) (lane 7) was used as controls.

Article Snippet: We incubated the cell lysates with 2 μg of anti-FBP1 antibody (FBP1 C-20; Santa Cruz Biotechnology) for 1 h at 4°C, then added 20 μl of protein A/G Plus agarose beads (Santa Cruz Biotechnology).

Techniques: Expressing, Stable Transfection, Knockdown, shRNA, Transfection, Plasmid Preparation, Clone Assay, Selection, Western Blot, Transformation Assay, Transduction, Control, Mutagenesis, Sequencing

Journal: BMC Cancer

Article Title: Fuse binding protein antagonizes the transcription activity of tumor suppressor protein p53

doi: 10.1186/1471-2407-14-925

Figure Lengend Snippet: FBP interacts with p53 and its regulatory proteins, and regulates their expression. (A) Effect of radiation-induced stress on the expression of p53, p21, BCCIP and TCTP in control and FBP-kd Huh 7 cells. Cells grown for 48 h were irradiated with 3-Gy of γ-ray. The irradiated cells were grown for 0, 3, 6, and 12 h and examined for the expression of p53, p21, BCCIP and TCTP by Western blotting. Lanes 1-4, show results from cells grown for 0, 3, 6 and 12 h post-irradiation (B) Fold change in the mRNA level of FBP1, p53, p21, BCCIP and TCTP in irradiated control and FBP-kd Huh7 cells. Cells grown for 48 h were irradiated with 3-Gy of γ-ray and grown for 0, 3, 6, and 12 h. Fold changes in mRNA level of FBP1, p53, p21, BCCIP, and TCTP were determined by real-time quantitative PCR. Upper and lower panels show fold change in mRNA level in irradiated control and FBP-kd cells, respectively. Lanes 1-4, show results from cells grown for 0, 3, 6 and 12 h post irradiation. (C, D) Co-immunoprecipitation of FBP1, p53 and BBCIP. IP was done with either (C) anti-FBP1 antibody or (D) anti-BCCIP antibody, and the IP complexes were captured on protein A/G PLUS-agarose beads, resolved on 8% polyacrylamide SDS-PAGE gel and Western blotted for p53, BCCIP and TCTP and FBP1. (E) FBP, p53 and BCCIP form ternary complex in-vitro. We incubated 500 ng of each of the recombinant FBP, p53 and BCCIP at 4 o C for 30 min. The complex was then subjected to FBP-IP, p53-IP or BCCIP-IP. The IP complex was resolved by SDS-PAGE and Western blotted for FBP, p53 and BCCIP. Lanes 1-3, show ternary complex of FBP1-p53-BCCIP captured by immunoprecipitation of FBP, p53 and BCCIP. Lane 4-6 show the formation of binary complexes between FBP1-p53, p53-BCCIP and FBP1-BCCIP, respectively.

Article Snippet: We incubated the cell lysates with 2 μg of anti-FBP1 antibody (FBP1 C-20; Santa Cruz Biotechnology) for 1 h at 4°C, then added 20 μl of protein A/G Plus agarose beads (Santa Cruz Biotechnology).

Techniques: Expressing, Control, Irradiation, Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, SDS Page, In Vitro, Incubation, Recombinant

Journal: BMC Cancer

Article Title: Fuse binding protein antagonizes the transcription activity of tumor suppressor protein p53

doi: 10.1186/1471-2407-14-925

Figure Lengend Snippet: The transactivation activity of p53 is activated in FBP-kd Huh7 cells. (A) Activation of p53 transcription activity in FBP-kd Huh7 cells. Reporter vectors were transfected to control Huh 7 cells or FBP-kd cells as described in the Methods. Forty-eight hours later, cells were irradiated with 4 Gy of γ-irradiation. Luciferase activities were measured 6-hour post-irradiation treatment. p53 reporter activity (left panel), p21 reporter activity (right panel). (B) Transient expression of FBP1 in FBP-kd cells suppresses p53 transcription activities. FBP-kd cells were transfected with shRNA resistant FBP1 expression clone (pCIA-CMV-FBP SHR ) along with reporter vectors; 48-h later cells were irradiated with 3 Gy of γ-irradiation. Luciferase activities were measured 6-hour post-irradiation treatment. p53 reporter activity (left panel), p21 reporter activity (right panel). (C) Relative p53 transcription activity in BCCIP-kd, p53-kd and FBP-kd cells. Huh7 cells knockdown for BCCIP; p53 and FBP1 were transfected with reporter vectors; 48-h later cells were irradiated with 3 Gy of γ-irradiation. Luciferase activities were measured 6-hour post-irradiation treatment. p53 reporter activity (left panel), p21 reporter activity (right panel).

Article Snippet: We incubated the cell lysates with 2 μg of anti-FBP1 antibody (FBP1 C-20; Santa Cruz Biotechnology) for 1 h at 4°C, then added 20 μl of protein A/G Plus agarose beads (Santa Cruz Biotechnology).

Techniques: Activity Assay, Activation Assay, Transfection, Control, Irradiation, Luciferase, Expressing, shRNA, Knockdown

Journal: Diabetologia

Article Title: CK2 activity is crucial for proper glucagon expression

doi: 10.1007/s00125-024-06128-1

Figure Lengend Snippet: Effect of CK2 downregulation on blood glucose, hormone secretion and FBP1 expression in mice. ( a ) Schematic illustration of the experimental setting. On day 0, αTC1 cells (WT or KO) were transplanted under the left kidney capsule of mice. Sham-transplanted mice served as negative control. Fasting blood glucose levels and body weights were measured over 28 days twice a week. On day 28, IPGTT was performed. ( b ) Body weight of mice transplanted with WT or KO cells from day 0 to day 28 ( n =6 each). Sham-transplanted mice served as negative control ( n =5 each). ( c ) AUC of the body weights shown in ( b ). ( d ) Blood glucose levels of mice transplanted with WT or KO cells from day 0 to day 28 ( n =6 each). Sham-transplanted mice served as negative control ( n =5 each). ( e ) AUC of the blood glucose levels shown in ( d ). ( f ) Quantitative analysis of blood glucose levels on day 28 according to the IPGTT of mice transplanted with WT or KO cells ( n =6 each). Sham-transplanted mice served as negative control ( n =5 each). ( g ) AUC of IPGTT results shown in ( f ). ( h ) Quantitative analysis of GCG secretion of mice transplanted with WT or KO cells ( n =8 each). Sham-transplanted mice served as negative control ( n =8 each). ( i ) Quantitative analysis of insulin secretion of mice transplanted with WT or KO cells ( n =3 each). Sham-transplanted mice served as negative control ( n =3 each). ( j ) Schematic illustration of the experimental setting. Mice were treated for 3 days with CX-4945 or DMSO (control) and blood, kidney and liver samples were collected to study GCG and insulin secretion and FBP1 expression. ( k ) Quantitative analysis of GCG secretion of mice treated with CX-4945 or DMSO (control) ( n =3 each). ( l ) Quantitative analysis of insulin secretion of mice treated with CX-4945 or DMSO (control) ( n =3 each). ( m ) Representative western blots of FBP1 and α-tubulin expression from liver and kidney tissue extracts. ( n , o ) Quantitative analysis of FBP1 from data shown in ( m ) ( n =2 or 3 each). Data are shown as mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001. ( a , j ) Generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. Ctrl, control (DMSO); CX, CX-4945; INS, insulin

Article Snippet: Fructose 1,6-bisphosphatase (FBP1) was detected with a monoclonal rabbit antibody (D2T7F, catalogue no. 59172, Cell Signaling Technology).

Techniques: Expressing, Negative Control, Control, Western Blot, Generated

Journal: Cell reports

Article Title: Branched-chain keto acids inhibit mitochondrial pyruvate carrier and suppress gluconeogenesis in hepatocytes

doi: 10.1016/j.celrep.2023.112641

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal to FBP1 , Cell Signaling Technology , # 72736; RRID: AB_2799827.

Techniques: Recombinant, Bioassay, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Bicinchoninic Acid Protein Assay, Cloning, Subcloning, Software

Journal: Cell metabolism

Article Title: Fructose-1,6-bisphosphatase is a nonenzymatic safety valve that curtails AKT activation to prevent insulin hyperresponsiveness

doi: 10.1016/j.cmet.2023.03.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The intensity of fluorescent-labeled protein-incorporated OPP was quantified by automated analysis of microscopic images with Image J software (NIH). . Generation and infection by AAV8-FBP1, AAV8-FBP1 E98A and AAV8-Ctrl virus pAAV[Exp]-CAG>mFbp1WT:T2A:EGFP:WPRE and pAAV[Exp]CAG>HA/mFbp1 E98A :T2A: EGFP:WPRE were constructed by Vectorbuilder.

Techniques: Immunoprecipitation, Transfection, Extraction, Enzyme-linked Immunosorbent Assay, ATP Assay, Colorimetric Assay, Activity Assay, Glucose Assay, In Situ, cDNA Synthesis, Recombinant, Sequencing, Construct, Software