wnt3 producing cell line  (ATCC)

Journal: World Journal of Gastrointestinal Oncology

Article Title: N-glycosylation of Wnt3 regulates the progression of hepatocellular carcinoma by affecting Wnt/β-catenin signal pathway

doi: 10.4251/wjgo.v16.i6.2769

Figure Lengend Snippet: Wnt3 is upregulated in hepatocellular carcinoma and modified by N-glycosylation at residues 90 and 301. A: According to the TCGA database query, the expression level of Wnt3 increased during the early stage of hepatocellular carcinoma; B: DEN and TAA were used to induce primary liver cancer in C3H mice, and normal mouse liver tissue was used as a control. The expression of Wnt3 in the two tissues was detected by immunohistochemical staining; C and H: Wnt3-GFP was transfected into PLC/PRF/5 cells for 36 h, which were then treated with TM for 24 h at a working concentration of 2.5 μg/mL; D and I: PLC/PRF/5 cells were transfected with Wnt3-GFP for 48 h, after which the cells were collected. Wnt3 was purified with a GFP tag, and the purified protein was treated according to the instructions of the PNGase F deglycosylation kit; E: Query of conserved areas of Wnt3 identified through the National Center for Biotechnology Information database; F: Schematic diagram showing the construction of site-directed Wnt3 mutants; G: Forty-eight hours after transfection, the cells were collected and subjected to Western blotting. LIHC: Liver hepatocellular carcinoma; GFP: Green fluorescent protein.

Article Snippet: The following antibodies were used: GAPDH mouse monoclonal antibody (P04406, Affinity, United States), GFP-tag (3A10) mouse monoclonal antibody (AP0675M, Bioworld, United States), RFP-tag mouse monoclonal antibody (MB2015, Bioworld, United States), Wnt3 mouse monoclonal antibody (67452-1-lg, Proteintech, United States), Frizzled-7 (Y87) rabbit polyclonal antibody (BS2774, Bioworld, United States), phospho-β-catenin (Ser552) rabbit polyclonal antibody (AF5749, Beyotime, China), and β-catenin rabbit polyclonal antibody (AF5126, Beyotime, China).

Techniques: Modification, Glycoproteomics, Expressing, Control, Immunohistochemical staining, Staining, Transfection, Concentration Assay, Purification, Western Blot

Journal: World Journal of Gastrointestinal Oncology

Article Title: N-glycosylation of Wnt3 regulates the progression of hepatocellular carcinoma by affecting Wnt/β-catenin signal pathway

doi: 10.4251/wjgo.v16.i6.2769

Figure Lengend Snippet: N-glycosylation affects the stability of Wnt3 and its ability to bind to the FZD7 receptor. A: The medium was replaced with serum-free medium 36 h after the transfection of PLC/PRF/5 cells. Twenty-four hours later, the medium was collected, ultrafiltration tubes were used for concentration, and then the samples were prepared for detection; B: At 36 h after the transfection of PLC/PRF/5 cells, actinomycin D was added, and the working concentration was 20 μg/mL; C: Localization of Wnt3 and its N-glycosylation deletion mutants (green) and FZD7 (red) in PLC/PRF/5 cells. The nuclei were stained with DAPI (blue); D and E: In PLC/PRF/5 and MHCC97H cells, anti-RFP was used for pull-down, and anti-GFP was used for detection. GFP: Green fluorescent protein; RFP: Red fluorescent protein; IP: Immunoprecipitation; IB: Immunoblotting.

Article Snippet: The following antibodies were used: GAPDH mouse monoclonal antibody (P04406, Affinity, United States), GFP-tag (3A10) mouse monoclonal antibody (AP0675M, Bioworld, United States), RFP-tag mouse monoclonal antibody (MB2015, Bioworld, United States), Wnt3 mouse monoclonal antibody (67452-1-lg, Proteintech, United States), Frizzled-7 (Y87) rabbit polyclonal antibody (BS2774, Bioworld, United States), phospho-β-catenin (Ser552) rabbit polyclonal antibody (AF5749, Beyotime, China), and β-catenin rabbit polyclonal antibody (AF5126, Beyotime, China).

Techniques: Glycoproteomics, Transfection, Concentration Assay, Staining, Immunoprecipitation, Western Blot

Journal: World Journal of Gastrointestinal Oncology

Article Title: N-glycosylation of Wnt3 regulates the progression of hepatocellular carcinoma by affecting Wnt/β-catenin signal pathway

doi: 10.4251/wjgo.v16.i6.2769

Figure Lengend Snippet: Quantification of the splice variants of Wnt3 through the HPA database

Article Snippet: The following antibodies were used: GAPDH mouse monoclonal antibody (P04406, Affinity, United States), GFP-tag (3A10) mouse monoclonal antibody (AP0675M, Bioworld, United States), RFP-tag mouse monoclonal antibody (MB2015, Bioworld, United States), Wnt3 mouse monoclonal antibody (67452-1-lg, Proteintech, United States), Frizzled-7 (Y87) rabbit polyclonal antibody (BS2774, Bioworld, United States), phospho-β-catenin (Ser552) rabbit polyclonal antibody (AF5749, Beyotime, China), and β-catenin rabbit polyclonal antibody (AF5126, Beyotime, China).

Techniques:

Journal: World Journal of Gastrointestinal Oncology

Article Title: N-glycosylation of Wnt3 regulates the progression of hepatocellular carcinoma by affecting Wnt/β-catenin signal pathway

doi: 10.4251/wjgo.v16.i6.2769

Figure Lengend Snippet: Site-directed mutations in Wnt3 affect signaling pathways downstream. A: The effect of different Wnt3 N-glycosylation-deficient mutants on the expression of effector molecules in the Wnt/β-catenin signaling pathway was assessed by Western blot analysis; B: ImageJ software was used to detect the IntDen values, and the p-β-catenin/GAPDH values were compared between the groups; C: ImageJ software was used to detect the IntDen values, and the Cyclin D1/GAPDH values were compared between the groups; D: ImageJ software was used to detect the IntDen values, and the C-myc/GAPDH values were compared between the groups; E: ImageJ software was used to detect the IntDen values, and the CDC42/GAPDH values were compared between the groups; F: ImageJ software was used to detect the IntDen values, and the Survivin/GAPDH values were compared between the groups. The data are presented as the mean ± SD from three independent experiments. a P < 0.05, b P < 0.01. GFP: Green fluorescent protein.

Article Snippet: The following antibodies were used: GAPDH mouse monoclonal antibody (P04406, Affinity, United States), GFP-tag (3A10) mouse monoclonal antibody (AP0675M, Bioworld, United States), RFP-tag mouse monoclonal antibody (MB2015, Bioworld, United States), Wnt3 mouse monoclonal antibody (67452-1-lg, Proteintech, United States), Frizzled-7 (Y87) rabbit polyclonal antibody (BS2774, Bioworld, United States), phospho-β-catenin (Ser552) rabbit polyclonal antibody (AF5749, Beyotime, China), and β-catenin rabbit polyclonal antibody (AF5126, Beyotime, China).

Techniques: Protein-Protein interactions, Glycoproteomics, Expressing, Western Blot, Software

Journal: World Journal of Gastrointestinal Oncology

Article Title: N-glycosylation of Wnt3 regulates the progression of hepatocellular carcinoma by affecting Wnt/β-catenin signal pathway

doi: 10.4251/wjgo.v16.i6.2769

Figure Lengend Snippet: Loss of N-glycosylation from Wnt3 weakens cell proliferation, invasion, migration and colony formation. A and B: Wound healing experiments were performed 36 h after PLC/PRF/5 cell transfection; C and D: At 36 h after transfection, the MHCC97H cells were subjected to wound healing experiments; E and F: At 36 h following the transfection of PLC/PRF/5 cells, after the cell count, 500 cells were removed for colony formation experiments; G and H: At 36 h after MHCC97H cell transfection, after the cell count, 500 cells were removed for colony formation experiments; I: Real-time cellular analysis (RTCA) was used to detect the proliferation of PLC/PRF/5 cells every 20 minutes for a total of 80 h; J: RTCA was used to detect the invasive ability of PLC/PRF/5 cells every 15 min for a total of 60 h; K: RTCA was used to detect the proliferation of MHCC97H cells every 20 min for a total of 80 h. a P < 0.01.

Article Snippet: The following antibodies were used: GAPDH mouse monoclonal antibody (P04406, Affinity, United States), GFP-tag (3A10) mouse monoclonal antibody (AP0675M, Bioworld, United States), RFP-tag mouse monoclonal antibody (MB2015, Bioworld, United States), Wnt3 mouse monoclonal antibody (67452-1-lg, Proteintech, United States), Frizzled-7 (Y87) rabbit polyclonal antibody (BS2774, Bioworld, United States), phospho-β-catenin (Ser552) rabbit polyclonal antibody (AF5749, Beyotime, China), and β-catenin rabbit polyclonal antibody (AF5126, Beyotime, China).

Techniques: Glycoproteomics, Migration, Transfection, Cell Counting

Journal: Clinical science (London, England : 1979)

Article Title: Restoration of the gut barrier integrity and restructuring of the gut microbiome in aging by angiotensin-(1–7)

doi: 10.1042/CS20220904

Figure Lengend Snippet: List of antibodies used for western blotting and immunohistochemistry

Article Snippet: Antibody Catalog number Vendor Concentration Claudin 1 (Host - Ms) 37-4900 Thermofisher 1:100 Occludin (Host -Rb) 40-4700 Thermofisher 1:100 Lgr5 (Host – Ms) MA5-25644 Thermofisher 1:100 Olfm4 (Host – Rb) PA5-115687 Thermofisher 1:50 F4/80 (Host - Rat) 14-4801-82 Invitrogen 1:50 CD11b (Host - Rb) ab133357 Abcam 1:4000 633 Goat Anti Rb 20122 Biotium 1:250 633 Goat Anti Ms 20010 Biotium 1:250 488 Goat Anti Rat A11006 Invitrogen 1:100 DAPI 40043 Biotium - ACE MA5- 32741 Thermofisher 1: 1000 ACE-2 MA5- 32307 Thermofisher 1: 1000 MAS1 (G-1) Sc- 390453 Santacruz 1: 1000 AT-1 (G-3) Sc- 515884 Santacruz 1: 1000 AT-2 MA5- 32293 Thermofisher 1: 1000 Wnt3a TA801736S Origene 1:1000 (western blot) and 1:100 (IHC) Open in a separate window List of antibodies used for western blotting and immunohistochemistry.

Techniques: Western Blot, Concentration Assay

Journal: Clinical science (London, England : 1979)

Article Title: Restoration of the gut barrier integrity and restructuring of the gut microbiome in aging by angiotensin-(1–7)

doi: 10.1042/CS20220904

Figure Lengend Snippet: (A) Shown were representative fluorescence images of Wnt3a immunohistochemistry with nuclear counter stain DAPI in colon sections derived from young, old and Ang-(1–7)-treated mice. (B) The number of Wnt3a-positive cells/villus was lower in the sections of old colon (n = 28 villi/5 mice/group) compared with the young (****P<0.0001, n = 30 villi/5 mice/group), which was increased in the Ang-(1–7)-treated old group (***P<0.001, n = 25 villi/5 mice/group). (C) Shown were representative Western blots of Wnt3a in the lysates of colon tissue or ex vivo-cultured organoids from different treatment groups. (D) Wnt3a protein levels were lower in the colons of old compared with the young (*P<0.05, n = 5 mice/group), which was increased in the colons derived from Ang-(1–7)-treated old group (*P<0.05, n = 5 mice/group). In the ex vivo-cultured organoids, decreased Wnt3a levels in the organoids from old compared with that derived from young colons, which was increased by treatment with Ang-(1–7) (*P<0.05, n = 5 mice/group). Data sets were analyzed by Kruskall–Wallis test followed by Dunn’s test.

Article Snippet: Antibody Catalog number Vendor Concentration Claudin 1 (Host - Ms) 37-4900 Thermofisher 1:100 Occludin (Host -Rb) 40-4700 Thermofisher 1:100 Lgr5 (Host – Ms) MA5-25644 Thermofisher 1:100 Olfm4 (Host – Rb) PA5-115687 Thermofisher 1:50 F4/80 (Host - Rat) 14-4801-82 Invitrogen 1:50 CD11b (Host - Rb) ab133357 Abcam 1:4000 633 Goat Anti Rb 20122 Biotium 1:250 633 Goat Anti Ms 20010 Biotium 1:250 488 Goat Anti Rat A11006 Invitrogen 1:100 DAPI 40043 Biotium - ACE MA5- 32741 Thermofisher 1: 1000 ACE-2 MA5- 32307 Thermofisher 1: 1000 MAS1 (G-1) Sc- 390453 Santacruz 1: 1000 AT-1 (G-3) Sc- 515884 Santacruz 1: 1000 AT-2 MA5- 32293 Thermofisher 1: 1000 Wnt3a TA801736S Origene 1:1000 (western blot) and 1:100 (IHC) Open in a separate window List of antibodies used for western blotting and immunohistochemistry.

Techniques: Fluorescence, Immunohistochemistry, Staining, Derivative Assay, Western Blot, Ex Vivo, Cell Culture

Journal: Clinical science (London, England : 1979)

Article Title: Restoration of the gut barrier integrity and restructuring of the gut microbiome in aging by angiotensin-(1–7)

doi: 10.1042/CS20220904

Figure Lengend Snippet: List of antibodies used for western blotting and immunohistochemistry

Article Snippet: Wnt3a , TA801736S , Origene , 1:1000 (western blot) and 1:100 (IHC).

Techniques: Western Blot, Concentration Assay

Journal: Clinical science (London, England : 1979)

Article Title: Restoration of the gut barrier integrity and restructuring of the gut microbiome in aging by angiotensin-(1–7)

doi: 10.1042/CS20220904

Figure Lengend Snippet: (A) Shown were representative fluorescence images of Wnt3a immunohistochemistry with nuclear counter stain DAPI in colon sections derived from young, old and Ang-(1–7)-treated mice. (B) The number of Wnt3a-positive cells/villus was lower in the sections of old colon (n = 28 villi/5 mice/group) compared with the young (****P<0.0001, n = 30 villi/5 mice/group), which was increased in the Ang-(1–7)-treated old group (***P<0.001, n = 25 villi/5 mice/group). (C) Shown were representative Western blots of Wnt3a in the lysates of colon tissue or ex vivo-cultured organoids from different treatment groups. (D) Wnt3a protein levels were lower in the colons of old compared with the young (*P<0.05, n = 5 mice/group), which was increased in the colons derived from Ang-(1–7)-treated old group (*P<0.05, n = 5 mice/group). In the ex vivo-cultured organoids, decreased Wnt3a levels in the organoids from old compared with that derived from young colons, which was increased by treatment with Ang-(1–7) (*P<0.05, n = 5 mice/group). Data sets were analyzed by Kruskall–Wallis test followed by Dunn’s test.

Article Snippet: Wnt3a , TA801736S , Origene , 1:1000 (western blot) and 1:100 (IHC).

Techniques: Fluorescence, Immunohistochemistry, Staining, Derivative Assay, Western Blot, Ex Vivo, Cell Culture