Journal: NPJ Precision Oncology

Article Title: FUT3 mediated GRP78 fucosylation promotes colorectal cancer survival and proliferation under glucose restriction via PERK/ATF4/STC2 axis

doi: 10.1038/s41698-025-01216-w

Figure Lengend Snippet: A The CCK-8 assay was conducted to investigate the effect of glucose restriction on the proliferation of SW480/Vector, SW480/FUT3 oe ; LOVO/Vector, LOVO/FUT3 oe ; SW480/NC, SW480/siFUT3; HCT116/NC, HCT116/siFUT3 groups. B The colony-forming assay was performed to assess the impact of glucose restriction on the proliferation of SW480/Vector, SW480/FUT3 oe ; LOVO/Vector, LOVO/FUT3 oe ; SW480/NC, SW480/siFUT3; HCT116/NC, HCT116/siFUT3 groups. C The multicellular spheroid assay was carried out to investigate the effect of glucose restriction on the proliferation of SW480/Vector, SW480/FUT3 oe ; LOVO/Vector, LOVO/FUT3 oe ; SW480/NC, SW480/siFUT3; HCT116/NC, HCT116/siFUT3 groups. D Flow cytometry was utilized to analyze the effect of glucose restriction on the cell cycle of SW480/Vector, SW480/FUT3 oe ; LOVO/Vector, LOVO/FUT3 oe ; SW480/NC, SW480/siFUT3; HCT116/NC, HCT116/siFUT3 groups. E The nude mice xenograft model was used to evaluate the effect of FUT3 on the proliferation of CRC cells in vivo. F Bar chart of the subcutaneous tumor volume. G Immunohistochemistry staining was performed to visualize the expression of FUT3 and Ki67 in tumors. H A bar chart was used to indicate the expression levels of FUT3 and Ki67 in tumors. I Western blot analysis was used to determine the expression levels of FUT3, ATF4, and STC2 in subcutaneous tumors.

Article Snippet: The tissue sections were incubated with antibodies against FUT3 (1:500; 67344-1-Ig, proteintech); ATF4 (1:200; 10835-1-AP, proteintech); STC2 (1:200; 60063-1-Ig, proteintech) and Ki-67 (1:4000; 27309-1-AP; proteintech), followed by probing with a secondary antibody and mounting with Diaminobenzidine (DAB).

Techniques: CCK-8 Assay, Plasmid Preparation, Flow Cytometry, In Vivo, Immunohistochemistry, Staining, Expressing, Western Blot

Journal: NPJ Precision Oncology

Article Title: FUT3 mediated GRP78 fucosylation promotes colorectal cancer survival and proliferation under glucose restriction via PERK/ATF4/STC2 axis

doi: 10.1038/s41698-025-01216-w

Figure Lengend Snippet: A Design scheme of GRP78 exon truncation mutations. B Validation of GRP78 exon truncation mutation efficiency. C Co-immunoprecipitation (coIP) assay demonstrates that FUT3 primarily binds to exon 8 of the GRP78 molecule. D The optimal parameter (lambda) for LASSO regression was selected based on ER stress-related genes. E LASSO coefficient profiles for ER stress-related genes in CRC were obtained. F AUC curves were used to evaluate the prognostic signature of ER stress-related genes. G A forest plot of multivariate Cox regression was used to analyze age, gender, AJCC stage, TNM stage, and risk score. H The expression of nine hub genes was compared between the low and high-risk groups. I The mRNA expression levels of FUT3 and STC2 were correlated in CRC tissues ( n = 38). J Immunohistochemistry staining was performed to explore the correlation of protein expression of FUT3 and STC2 in CRC tissues.

Article Snippet: The tissue sections were incubated with antibodies against FUT3 (1:500; 67344-1-Ig, proteintech); ATF4 (1:200; 10835-1-AP, proteintech); STC2 (1:200; 60063-1-Ig, proteintech) and Ki-67 (1:4000; 27309-1-AP; proteintech), followed by probing with a secondary antibody and mounting with Diaminobenzidine (DAB).

Techniques: Biomarker Discovery, Mutagenesis, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Immunohistochemistry, Staining

Journal: NPJ Precision Oncology

Article Title: FUT3 mediated GRP78 fucosylation promotes colorectal cancer survival and proliferation under glucose restriction via PERK/ATF4/STC2 axis

doi: 10.1038/s41698-025-01216-w

Figure Lengend Snippet: A The correlation between the ATF4 and STC2 was explored via the GEPIA. B CO-IP was used to detect the binding of GRP78 and PERK with SW480/Vector and SW480/FUT3 oe groups. C The qRT-PCR results of ATF4 and STC2 were obtained from SW480/Vector, SW480/FUT3 oe , HCT116/NC, and HCT116/siFUT3 groups under high (25 mM) or restricted (2.5 mM) glucose conditions. D Western blot results were obtained for FUT3, GRP78, PERK, p-PERK, ATF4, and STC2 from SW480/Vector, SW480/FUT3 oe , HCT116/NC, and HCT116/siFUT3 groups under high (25 mM) or restricted (2.5 mM) glucose conditions. E CoIP and AAL lectin assays confirm that SGN-2FF specifically inhibits GRP78 fucosylation and reduces PERK dissociation from GRP78. F Western blot analysis of the effect of SGN-2FF-mediated inhibition of GRP78 fucosylation on downstream signaling pathway activation. G Cell cycle assay examining the rescue effect of SGN-2FF on CRC proliferation. H Colony formation assay evaluating the rescue effect of SGN-2FF on CRC proliferation. I CCK8 assay investigating the rescue effect of SGN-2FF on colorectal cancer (CRC) proliferation. J Flow cytometry apoptosis staining assessing the rescue effect of SGN-2FF on CRC apoptosis.

Article Snippet: The tissue sections were incubated with antibodies against FUT3 (1:500; 67344-1-Ig, proteintech); ATF4 (1:200; 10835-1-AP, proteintech); STC2 (1:200; 60063-1-Ig, proteintech) and Ki-67 (1:4000; 27309-1-AP; proteintech), followed by probing with a secondary antibody and mounting with Diaminobenzidine (DAB).

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Inhibition, Activation Assay, Cell Cycle Assay, Colony Assay, CCK-8 Assay, Flow Cytometry, Staining

Journal: NPJ Precision Oncology

Article Title: FUT3 mediated GRP78 fucosylation promotes colorectal cancer survival and proliferation under glucose restriction via PERK/ATF4/STC2 axis

doi: 10.1038/s41698-025-01216-w

Figure Lengend Snippet: A The expression of FUT3 and ATF4 was analyzed in SW480 and HCT116 cells with FUT3 overexpression and knockdown under restricted glucose conditions using nuclear and cytoplasmic separation assays. B The ATF4-binding motif on STC2 was identified using JASPAR. C and D ATF4 binding to predicted sites on STC2 was confirmed by ChIP-qPCR and DNA agarose gel electrophoresis using anti-ATF4, control IgG antibodies, and Histone H3 antibodies. E The luciferase reporter assay was used to determine that the ATF4 binding site (+35 bp) was critical in modulating STC2 transcriptional activity. F An immunofluorescence assay was used to detect the expression and nuclear import of ATF4 under 2.5 mM glucose conditions (WT: Control plasmid encoding firefly luciferase, MUT: Plasmid of interest containing the STC2 promoter upstream of firefly luciferase, Vector: Empty vector control plasmid, ATF4oe: ATF4 overexpression plasmid).

Article Snippet: The tissue sections were incubated with antibodies against FUT3 (1:500; 67344-1-Ig, proteintech); ATF4 (1:200; 10835-1-AP, proteintech); STC2 (1:200; 60063-1-Ig, proteintech) and Ki-67 (1:4000; 27309-1-AP; proteintech), followed by probing with a secondary antibody and mounting with Diaminobenzidine (DAB).

Techniques: Expressing, Over Expression, Knockdown, Binding Assay, ChIP-qPCR, Agarose Gel Electrophoresis, Control, Luciferase, Reporter Assay, Activity Assay, Immunofluorescence, Plasmid Preparation

Journal: NPJ Precision Oncology

Article Title: FUT3 mediated GRP78 fucosylation promotes colorectal cancer survival and proliferation under glucose restriction via PERK/ATF4/STC2 axis

doi: 10.1038/s41698-025-01216-w

Figure Lengend Snippet: A qPCR analysis of ATF4 and STC2 mRNA expression levels following PERK inhibitor treatment. B Western blot assessment of PERK/ATF4/STC2 pathway activation upon PERK inhibitor administration. C CCK-8 assay evaluating the rescue effect of PERK inhibition on CRC proliferative capacity. D Colony formation assay demonstrating the restorative effect of PERK inhibition on CRC proliferation. E 3D spheroid formation assay confirming the rescue of CRC proliferative capacity with PERK inhibitor treatment. F Flow cytometric apoptosis analysis showing the rescue of anti-apoptotic capacity by PERK inhibition. G qPCR validation of STC2 knockdown efficiency following siRNA transfection. H Western blot verification of STC2 knockdown efficiency. I CCK-8 assay examining the rescue effect of STC2 knockdown on CRC proliferation. J Colony formation assay demonstrating the restorative effect of STC2 interference on CRC proliferative capacity. K 3D spheroid formation assay confirming the rescue of CRC proliferation following STC2 knockdown. L Flow cytometric apoptosis analysis showing the restoration of anti-apoptotic capacity with STC2 interference.

Article Snippet: The tissue sections were incubated with antibodies against FUT3 (1:500; 67344-1-Ig, proteintech); ATF4 (1:200; 10835-1-AP, proteintech); STC2 (1:200; 60063-1-Ig, proteintech) and Ki-67 (1:4000; 27309-1-AP; proteintech), followed by probing with a secondary antibody and mounting with Diaminobenzidine (DAB).

Techniques: Expressing, Western Blot, Activation Assay, CCK-8 Assay, Inhibition, Colony Assay, Tube Formation Assay, Biomarker Discovery, Knockdown, Transfection