Journal: Cancer medicine

Article Title: IGF2BP2 promotes pancreatic carcinoma progression by enhancing the stability of B3GNT6 mRNA via m6A methylation.

doi: 10.1002/cam4.5096

Figure Lengend Snippet: FIGURE 5 IGF2BP2 regulates B3GNT6 mRNA expression to promote pancreatic carcinoma. (A) Prediction of IGF2BP2 target genes, Venn diagram shows substantial and significant overlap among TCGA-GTEx, IGF2BP2-CLIP and DEGs between PA1 and PA2 subgroups. (B) Expression of predicted genes after IGF2BP2 KO. C m6A IP and IGF2BP2 IP verified the binding efficacy of target genes. (D) TCGA- GTEx reveals the high expression of B3GNT6 in PC. (E) Spearman correlation analysis reveals positive co-expression between IGF2BP2 and B3GNT6. (F–H) CCK-8 and EdU assay revealed the PC cell proliferation ability after B3GNT6 KO. (I) Transwell experiment revealed PC cell migration ability after B3GNT6 KO. (J) B3GNT6 KO inhibited colony formation ability in PC cells. (*p < 0.05).

Article Snippet: After dewaxing, hydration and antigen repair, paraffin sections were blocked with immunostaining blocking solution for 1 h, incubated with IGF2BP2 and B3GNT6 (ID:21291- 1- AP, Proteintech, American) primary antibody at 4°C overnight and secondary antibody for 1 h. After counterstaining with DAPI, sections were sealed with an anti- fluorescence quenching agent and imaged under a fluorescence microscope.

Techniques: Expressing, Binding Assay, CCK-8 Assay, EdU Assay, Migration

Journal: Cancer medicine

Article Title: IGF2BP2 promotes pancreatic carcinoma progression by enhancing the stability of B3GNT6 mRNA via m6A methylation.

doi: 10.1002/cam4.5096

Figure Lengend Snippet: FIGURE 7 Mechanism diagram of IGF2BP2 promoting B3GNT6 expression via m6A methylation.

Article Snippet: After dewaxing, hydration and antigen repair, paraffin sections were blocked with immunostaining blocking solution for 1 h, incubated with IGF2BP2 and B3GNT6 (ID:21291- 1- AP, Proteintech, American) primary antibody at 4°C overnight and secondary antibody for 1 h. After counterstaining with DAPI, sections were sealed with an anti- fluorescence quenching agent and imaged under a fluorescence microscope.

Techniques: Expressing, Methylation

Journal: bioRxiv

Article Title: Restoring O-glycosylation and expression of MUC2 limits progression of colorectal cancer

doi: 10.1101/2024.01.25.577208

Figure Lengend Snippet: (A) GSEA was performed to analyze a specific enrichment of factors in the Mucin type O-glycan biosynthesis pathway. (B) PPI network was analyzed with GeneMANIA. (C-D) IHC staining and quantitation of B3GNT6 in the CRC patients. Scale bars, 50 µm. (E-F) Lectin staining using BSL-II and quantitation of Core3 in the CRC. (G) Co-IP analysis to detect interaction of MUC2 and B3GNT6 after transfection of pcDNA3.1-B3GNT6-Flag vector, the protein complex bound to B3GNT6 was pulled down by anti-flag antibody (n=3, Input; n=3, IgG; n=3, B3GNT6). (H) Co-IP and lectin staining analysis to detect the expression of Core3 in MUC2 after transfection of pcDNA3.1-B3GNT6-Flag vector, the protein complex was pulled down by MUC2 antibody (n=3, Input; n=3, IgG; n=3, B3GNT6). (I) StcE specifically cleaves MUC2 O-glycosylation modified peptides, resulting in MUC2 degradation. (J) Western Blot to detect the degradation of MUC2 after the LOVO cells were treated with different concentrations of StcE (0nM, 4nM, 8nM, 16nM, 32nM, 64nM) for 3 hours. Data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons. The error bars indicate means ±standard error (SEM). *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; PPI, the protein–protein interaction; GSEA, gene set enrichment analysis, IHC, immunohistochemistry; Co-IP, co-immunoprecipitation; StcE, Recombinant Escherichia coli O157: H7 Metalloprotease.

Article Snippet: Mix anti-Flag immunomagnetic beads with 4ug IgG antibody (Proteintech, B900620) or 4ug B3GNT6 antibody (Cusabio, CSB-PA744392LA01HU) and incubate on the DNA Mixer for 3 hours.

Techniques: Glycoproteomics, Immunohistochemistry, Quantitation Assay, Staining, Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Expressing, Modification, Western Blot, Immunoprecipitation, Recombinant

Journal: bioRxiv

Article Title: Restoring O-glycosylation and expression of MUC2 limits progression of colorectal cancer

doi: 10.1101/2024.01.25.577208

Figure Lengend Snippet: (A) Boxplot analysis of B3GNT6 expression level in tumors and normal tissues in CRC patients in TCGA. (B) A scatter plot and Pearson correlation score showing a positive correlation between MUC2 and B3GNT6 in TCGA. (C) Schematic diagram of the synthetic pathway of O-glycosylation. Data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons. (D-E) QPCR and Western Blot assay to evaluate the expression level of MUC2 after pcDNA3.1-B3GNT6 plasmid transfection into LOVO cells (n=3, Ctrl; n=3, B3GNT6-OE) The error bars indicate means ±standard error (SEM). *p<0.05, **p<0.01, ***p<0.001. Data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons. The error bars indicate means ±standard error (SEM). *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; qPCR, quantitative polymerase chain reaction.

Article Snippet: Mix anti-Flag immunomagnetic beads with 4ug IgG antibody (Proteintech, B900620) or 4ug B3GNT6 antibody (Cusabio, CSB-PA744392LA01HU) and incubate on the DNA Mixer for 3 hours.

Techniques: Expressing, Glycoproteomics, Western Blot, Plasmid Preparation, Transfection, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Restoring O-glycosylation and expression of MUC2 limits progression of colorectal cancer

doi: 10.1101/2024.01.25.577208

Figure Lengend Snippet: (A) Lectin staining using BSL-II and in LOVO cells after transfection of pcDNA3.1-B3GNT6 plasmid into LOVO cells (n=3, Ctrl; n=3, B3GNT6-OE). Scale bars, 50 µm. (B) MUC2 secretion was measured by ELISA after transfection of pcDNA3.1-B3GNT6 plasmid into LOVO cells (n=5, Ctrl; n=5, B3GNT6-OE). (C) ELSIA assay evaluated the level of MUC2 in cell lysate and cell supernatant after pcDNA3.1-B3GNT6 plasmid transfection into LOVO cells (n=5, Intracellular-Ctrl; n=5, Intracellular-B3GNT6; n-5, Secreted-Ctrl; n=5, Secreted-B3GNT6). Data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons. The error bars indicate means ±standard error (SEM). *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; OE, Overexpression; ELISA, enzyme-linked immunosorbent assay.

Article Snippet: Mix anti-Flag immunomagnetic beads with 4ug IgG antibody (Proteintech, B900620) or 4ug B3GNT6 antibody (Cusabio, CSB-PA744392LA01HU) and incubate on the DNA Mixer for 3 hours.

Techniques: Staining, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Over Expression

Journal: bioRxiv

Article Title: Restoring O-glycosylation and expression of MUC2 limits progression of colorectal cancer

doi: 10.1101/2024.01.25.577208

Figure Lengend Snippet: (A) Western Blot to detect the expression of MUC2 after the cells were treated with different concentrations of KLF4 inhibitor Kenpaullone (0uM, 0.5uM, 1uM, 2.5uM, 5uM) for 24 hours. (B-C) IHC staining and quantitation of KLF4 in the CRC patients (n=31, normal tissue; n=31, tumor tissue). Scale bars, 50 µm. (D-E) ChIP enrichment was quantified using qPCR and PCR analysis to detect MUC2-binding site (n=3, Input; n=3, IgG; n=3, CHIP-KLF4). (F) The above plasmids and pCMV-Renilla plasmid were co-transfected into LOVO cells, and the normalized luciferase activity was measured (n=5, Ctrl; n=5, WT; n=5, mut). (G-H) ChIP enrichment was quantified using qPCR and PCR analysis to detect B3GNT6-binding sites (n=3, Input; n=3, IgG; n=3, CHIP-KLF4). (I) The above plasmids and pCMV-Renilla plasmid were co-transfected into LOVO cells, and the normalized luciferase activity was measured (n=5, Ctrl; n=5, WT; n=5, mut1; n=5, mut2; n=5, mut12).Data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons. The error bars indicate means ±standard error (SEM). *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; qPCR, quantitative polymerase chain reaction; ChIP, chromatin immunoprecipitation; CRC, Colorectal Cancer.

Article Snippet: Mix anti-Flag immunomagnetic beads with 4ug IgG antibody (Proteintech, B900620) or 4ug B3GNT6 antibody (Cusabio, CSB-PA744392LA01HU) and incubate on the DNA Mixer for 3 hours.

Techniques: Western Blot, Expressing, Immunohistochemistry, Quantitation Assay, Binding Assay, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

Journal: bioRxiv

Article Title: Restoring O-glycosylation and expression of MUC2 limits progression of colorectal cancer

doi: 10.1101/2024.01.25.577208

Figure Lengend Snippet: (A) A scatter plot and Pearson correlation score showing a positive correlation between B3GNT6 and KLF4 in TCGA. (B-C) QPCR and Western Blot to detect the expression of B3GNT6 after transfection pcDNA3.1-KLF4 plasmid or siRNA-KLF4 into LOVO cells (n=3, Ctrl; n=3, KLF4-OE; n=3, siRNA-KLF4). (D) Schematic diagram of KLF4 binding site in the B3GNT6 promoter region. (E) Schematic diagram of B3GNT6 promoter reporter plasmids. PGL3basic-B3GNT6 promoter-WT plasmid was constructed by inserting B3GNT6 promoter sequence into pGLBasic luciferase reporter plasmid. pGL3basic-Mutated plasmids were constructed by mutating two binding sites separately or at the same time (PGL3basic-B3GNT6 promoter-mut1: 1924-1934bp; PGL3basic-B3GNT6 promoter-mut2: 155-165bp). Data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons. The error bars indicate means ±standard error (SEM). *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; qPCR, quantitative polymerase chain reaction.

Article Snippet: Mix anti-Flag immunomagnetic beads with 4ug IgG antibody (Proteintech, B900620) or 4ug B3GNT6 antibody (Cusabio, CSB-PA744392LA01HU) and incubate on the DNA Mixer for 3 hours.

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Binding Assay, Construct, Sequencing, Luciferase, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Restoring O-glycosylation and expression of MUC2 limits progression of colorectal cancer

doi: 10.1101/2024.01.25.577208

Figure Lengend Snippet: (A-B) IHC staining and quantitation of PPARg in the CRC patients (n=31, normal tissue; n=31, tumor tissue). Scale bars, 50 µm. (C-D) Western Blot to detect the expression levels of PPARg, MUC2, KLF4 and B3GNT6 after the cells were treated with different concentrations of Rosiglitazone (0, 10uM, 20uM, 40uM, 60uM) or GW9662 (0, 5uM, 10uM, 15uM, 20uM) for 24 hours. (E-F) ChIP enrichment was quantified using PCR and qPCR analysis to detect KLF4-binding site (n=3, Input; n=3, IgG; n=3, CHIP-PPARg). (G) The above plasmids and pCMV-Renilla plasmid were co-transfected into LOVO cells, and the normalized luciferase activity was measured (n=5, Ctrl; n=5, WT; n=5, mut). (H-I) Western Blot detect the expression levels of PPARg, KLF4 and MUC2 after the cells were treated with siRNA-KLF4 transfection and Rosiglitazone (40uM), or with pcDNA3.1-KLF4 plasmid transfection and GW9662 (20uM). Data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons. The error bars indicate means ±standard error (SEM). *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; IHC, immunohistochemistry; ChIP, chromatin immunoprecipitation; qPCR, quantitative polymerase chain reaction; CRC, Colorectal Cancer.

Article Snippet: Mix anti-Flag immunomagnetic beads with 4ug IgG antibody (Proteintech, B900620) or 4ug B3GNT6 antibody (Cusabio, CSB-PA744392LA01HU) and incubate on the DNA Mixer for 3 hours.

Techniques: Immunohistochemistry, Quantitation Assay, Western Blot, Expressing, Binding Assay, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Restoring O-glycosylation and expression of MUC2 limits progression of colorectal cancer

doi: 10.1101/2024.01.25.577208

Figure Lengend Snippet: (A) ELSIA assay evaluated the level of MUC2 in cell supernatant after treated with Rosiglitazone (40uM) or GW9662 (20uM) (n=5, Ctrl; n-5, Rosiglitazone; n=5, GW9662). (B) H&E, IHC staining of PPARg, KLF4, B3GNT6, MUC2 and BSL-II lectin staining of Core3 in the C57BL/6 treated with Rosiglitazone (10mg/kg) or GW9662 (10mg/kg) (n=3, Ctrl; n=3, Rosiglitazone; n=3, GW9662). Scale bars, 50 µm. Data were analyzed by ordinary one-way ANOVA with Tukey’s multiple comparisons. The error bars indicate means ±standard error (SEM). *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; H&E, hematoxylin-eosin; IHC, immunohistochemistry; CRC, Colorectal Cancer.

Article Snippet: Mix anti-Flag immunomagnetic beads with 4ug IgG antibody (Proteintech, B900620) or 4ug B3GNT6 antibody (Cusabio, CSB-PA744392LA01HU) and incubate on the DNA Mixer for 3 hours.

Techniques: Immunohistochemistry, Staining