Journal: Scientific Reports

Article Title: A novel signaling pathway regulates colon cancer angiogenesis through Norrin

doi: 10.1038/srep05630

Figure Lengend Snippet: In column 1, low power (5×) micrographs of the tumor microenvironment are depicted. The square demonstrates the region of higher (100×) magnification (columns 2–5). Green fluorescence (column 2) indicates expression of a specific Norrin signaling component (see row designation, below). Red fluorescence (column 3) marks endothelial cells (anti-CD31 positive) of colorectal tumor blood vessels. Blue fluorescence(column 4) depicts DAPI-stained nuclei. Merged images are depicted in column 5. Row (A). Expression of Norrin in colorectal tumor endothelial cells. Row (B). Expression of Frizzled 4 in colorectal tumor endothelial cells. Row (C). Expression of Lrp5 in colorectal tumor endothelial cells. Row (D). Expression of TSPAN12 in colorectal tumor endothelial cells.

Article Snippet: Colorectal tumor slides were triple stained with DAPI (blue) for nuclear DNA, Texas Red (red) for human blood vessel endothelial cells (anti-CD31 mouse monoclonal antibody Abcam (ab 9498) at 1:10 dilution, and a secondary antibody conjugate Texas Red-donkey polyclonal against mouse IgG from Abcam (ab 7059) used at 1:1000 dilution) and FITC (green) for components of Norrin pathway (goat polyclonal directed against human Norrin (R&D, AF 3014), diluted till 1:50 from stock 0.2 mg/ml; rabbit polyclonal against human Frizzled 4 (MBL International Corporation, MC-4911), diluted 1:40; goat polyclonal against human Lrp 5 (Abcam, ab 36121), diluted 1:500; rabbit polyclonal against human TSPAN12 (Biorbyt, orb 39384), diluted at 1:100.

Techniques: Fluorescence, Expressing, Staining

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Primer sequences of TSPAN12.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Amplification

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Chromatograms and pedigrees of three families with familial exudative vitreoretinopathy. Three novel mutations were identified in TSPAN12 . A : In Family A, the affected mother and son had the c.566G>A (p.C189Y) mutation. C : In Family B, the patient and her affected mother had the c.177delC (p.Y59fsX67) mutation. E : In Family C, the affected mother and son had the c.C254T (p.T85M) mutation. The columns from left to right display the pedigree and the sequence chromatograms of these patients ( A , C , E ) and the normal controls ( B , D , F ). Arrows indicate the positions of the altered nucleotides.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Mutagenesis, Sequencing

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Protein sequence alignment of human TSPAN12 with its orthologs. The conserved amino acid residues are shaded. The orthologs are from the following species: Homo sapiens ( NP_036470 ), Pan troglodytes ( XP_001142754 ), Musmusculus ( NP_766595 ), Rattus norvegicus ( NP_001015026 ), Bos taurus ( NP_001039977 ), Equus caballus ( XP_001502093 ), Canis lupus familiaris ( XP_855095 ), Monodelphis domestica ( XP_001364876 ), Gallus gallus ( NP_001007850 ), Taeniopygia guttata ( XP_002192381 ), Ornithorhynchus anatinus ( XP_001516347 ), and Danio rerio (NP_957446). A : The residue of the missense mutation p.C189Y is highly conserved. B : The residue of the missense mutation p.T85M is also highly conserved.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Sequencing, Residue, Mutagenesis

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Luciferase assays with the SuperTopFlash cell line transfected with the indicated plasmids. SuperTopFlash (STF) cells/well were transfected with 800 ng DNA (200 ng of Norrin, 200 ng of FZD4, 200 ng of LRP5, 100 ng of pSV-β-galactosidase control vector, and 100 ng of TSPAN12 plasmid [wild-type or mutation]) and 1.5 µl Lipofectamine 2000 transfection reagent. Forty-eight hours after transfection, the cells were harvested and washed twice with PBS. Luciferase activities were measured with a dual-luciferase assay kit. Reporter activity was normalized to the coexpressed β-galactosidase activity in each well. Each test was performed in triplicate. The reporter assay was repeated three times, and a representative result was obtained.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Luciferase, Transfection, Control, Plasmid Preparation, Mutagenesis, Activity Assay, Reporter Assay

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Western blot analysis by SDS-PAGE of the TSPAN12 mutants. Total protein (10 μg) isolated from cell lysates from luciferase assays was mixed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer and subjected to SDS-PAGE and western blot analysis using anti-Flag antibody to detect TSPAN12 and FZD4 expression. Beta-actin was used as the loading control. The expression level of TSPAN12 C189Y was compatible with that of the wild-type. However, the T85M and Y59fs mutant proteins were not stable.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Western Blot, SDS Page, Isolation, Luciferase, Polyacrylamide Gel Electrophoresis, Expressing, Control, Mutagenesis

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Immunofluorescence staining of the TSPAN12 C189Y mutant. Cos 7 cells were transfected either with human wild-type or mutant TSPAN12 cloned into the pCMV6-entry vector, or empty vector. Cells were washed with PBS after 48 h and fixed with 4% PFA for 15 min. Mouse monoclonal anti-Flag antibody and Alexa Fluor 594 goat anti-mouse immunoglobulin (IgG) secondary antibody were used to detect TSPAN12 expression with the standard immunostaining method. Red channel, TSPAN12; blue channel, 4',6-diamidino-2-phenylindole (DAPI) for nuclei staining.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques: Immunofluorescence, Staining, Mutagenesis, Transfection, Clone Assay, Plasmid Preparation, Expressing, Immunostaining

Journal: Molecular Vision

Article Title: Novel mutations in the TSPAN12 gene in Chinese patients with familial exudative vitreoretinopathy

doi:

Figure Lengend Snippet: Diagram showing 9 known TSPAN12 mutations and three novel mutations identified in this study. The novel mutations are red.

Article Snippet: The cDNA encoding wild-type TSPAN12 (Origene, Rockville, MD) was subcloned in-frame into the pCMV-entryMyc-Flag vector (Origene) using SgfI and MluI sites.

Techniques:

Journal: bioRxiv

Article Title: The co-receptor Tspan12 directly captures Norrin to promote ligand-specific β-catenin signaling

doi: 10.1101/2024.02.03.578714

Figure Lengend Snippet: A. Schematic of BLI setup for Tspan12-Norrin binding: Tspan12, inserted into biotinylated nanodiscs, is immobilized on a streptavidin-coated biosensor, and Norrin association and dissociation are monitored in real time. B. BLI traces of Norrin at indicated concentrations binding to and dissociating from Tspan12. C. Steady-state binding curve fit to Norrin-Tspan12 binding (mean ± S.D. from three independent replicates at each concentration of Norrin) gives a K D of 10.4 ± 1.2 nM (mean ± S.E.M.) D. Observed association rate constant (K obs ), determined from fitting BLI association traces (mean ± S.D. in three independent experiments), is linearly dependent on Norrin concentration with a slope K on = 0.00019 ± 0.00003 nM -1 s -1 (mean ± S.E.M.). When combined with the K off = 0.0014 ± 0.00016 s -1 (mean ± S.E.M.) determined from fitting the dissociation traces, we obtain a kinetic K D of 7.4 ± 1.4 nM (mean ± S.E.M.) E. BLI trace of the soluble MBP-tagged Tspan12 LEL domain, at the indicated concentrations, associating to and dissociating from a biosensor loaded with MBP-tagged Norrin. Kinetic fitting gives an apparent affinity of 16 ± 3 nM (mean ± S.E.M.). F. BLI traces of 10, 32 or 100 nM Norrin show no binding to a biosensor loaded with a nanodisc-embedded chimeric Tspan12, with the LEL replaced by that of Tspan11.

Article Snippet: For expression in Sf9 cells, full-length human Tspan12 (DNASU), Tspan12ΔC (residues 1-252), and Tspan12 with the large extracellular loop replaced by that of TSPAN11 (Addgene Plasmid #115785 from Harald Junge ( )) were C-terminally tagged with the Rho-1D4 antibody recognition sequence TETSQVAPA.

Techniques: Binding Assay, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Increased CD40 expression on muscle cells of polymyositis and dermatomyositis: role of CD40-CD40 ligand interaction in IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 production.

doi: 10.4049/jimmunol.164.12.6593

Figure Lengend Snippet: FIGURE 1. Immunohistochemical staining with Abs against CD40 (A), CD40L (B), IFN-g (C), and control Ig (D and E) in muscle tissue of PM/ DM. A, mAb against CD40 labeled the sarcolemma of muscle fibers (as- terisk), as well as endomysial MNCs (thick arrow) and endothelial cells of the capillary wall (thin arrow) obtained from a patient with PM (patient 1). CD40-positive fibers tended to be smaller in diameter than CD40-negative fibers. Similar results were observed in patients 2–9. B, Infiltrating MNCs surrounding muscle cells expressed CD40L (patient 1). Similar results were observed in patients 2–10. C, MNCs in the endomysium as well as the perivascular area expressed IFN-g in a patient with DM (patient 6). Similar results were observed in patients 1–4 and 7–9. D, The staining of control mouse IgG1. E, The staining of control rabbit IgG. Original magnification, 3400.

Article Snippet: Briefly, all sections were incubated with 2.5% normal horse serum for 30 min, followed by mAbs recognizing CD40 (Mab89, 10 mg/ ml, mouse IgG1; Coulter, Hialeah, FL), CD40L (thrombin receptor agonist peptide 1 (TRAP1), 10 mg/ml, mouse IgG1; Coulter), IL-15 (10 mg/ml, mouse IgG1; R&D Systems, Minneapolis, MN), and MCP-1 (10 mg/ml, mouse IgG1; Chemicon, Temecula, CA) or polyclonal Abs for IFN-g (5 mg/ml, rabbit IgG; Serotec, Oxford, U.K.) and IL-6 (5 mg/ml, goat IgG; Dako, Glostrup, Denmark) for 30 min.

Techniques: Immunohistochemical staining, Staining, Control, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Increased CD40 expression on muscle cells of polymyositis and dermatomyositis: role of CD40-CD40 ligand interaction in IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 production.

doi: 10.4049/jimmunol.164.12.6593

Figure Lengend Snippet: FIGURE 3. A, CD40 mRNA expression in SkMC 2859. Total RNA was prepared from un- stimulated and IFN-g-stimulated SkMC 2859. cDNAs of CD40 and actin were amplified by RT-PCR. Product sizes for CD40 and b-actin genes are 430 and 218 bp, respectively. Lane 1, Unstimulated SkMC 2859; lane 2, IFN-g-stim- ulated SkMC 2859. B, Induction of CD40 ex- pression on SkMC 2859 by IFN-g and/or TNF-a. Upper panel, SkMC 2859 were stimu- lated with various concentrations of IFN-g for 48 h, stained with anti-CD40 mAb, and analyzed by flow cytometry (solid histogram). Fluores- cence levels for isotype-matched negative con- trol mAb are indicated by the dotted histogram. The x-axis represents the fluorescence intensity, and the y-axis shows the cell number. IFN-g in- duced CD40 expression on SkMC 2859 in a dose-dependent manner. Lower panel, Surface CD40 expression on resting or cytokine-stimu- lated SkMC 2859. SkMC 2859 were stimulated or unstimulated with TNF-a alone, IFN-g alone, or a combination of these cytokines for 48 h; stained with anti-CD40 Ab; and analyzed by flow cytometry (solid histogram). The back- ground fluorescence levels are shown by the dot- ted histogram. IFN-g and TNF-a showed a syn- ergistic effect on CD40 induction. p, Percentage of positive cells.

Article Snippet: Briefly, all sections were incubated with 2.5% normal horse serum for 30 min, followed by mAbs recognizing CD40 (Mab89, 10 mg/ ml, mouse IgG1; Coulter, Hialeah, FL), CD40L (thrombin receptor agonist peptide 1 (TRAP1), 10 mg/ml, mouse IgG1; Coulter), IL-15 (10 mg/ml, mouse IgG1; R&D Systems, Minneapolis, MN), and MCP-1 (10 mg/ml, mouse IgG1; Chemicon, Temecula, CA) or polyclonal Abs for IFN-g (5 mg/ml, rabbit IgG; Serotec, Oxford, U.K.) and IL-6 (5 mg/ml, goat IgG; Dako, Glostrup, Denmark) for 30 min.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Increased CD40 expression on muscle cells of polymyositis and dermatomyositis: role of CD40-CD40 ligand interaction in IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 production.

doi: 10.4049/jimmunol.164.12.6593

Figure Lengend Snippet: FIGURE 4. A, Effect of rhCD40L on cytokine production of SkMC 2859. SkMC 2859 were stimulated or unstimulated with 100 U/ml of IFN-g for 48 h to induce CD40 expression, and then cultured in medium containing various concentrations of rhCD40L for 48 h. At the end of culture, the culture supernatants and cell extracts were collected, and the concentrations of cytokines were determined by ELISA. The results are expressed as the mean 6 SE (n 5 3). f, without IFN-g, intracellular cytokines; M, without IFN-g, secreted cytokines; p, stimulated with IFN-g, intracellular cytokines; u, stimulated with IFN-g, secreted cytokines. p, p , 0.05; pp, p , 0.01. ND, not detectable. B, mRNA expression of cytokine in SkMC 2859. Total RNA was prepared from rhCD40L-stimulated SkMC 2859 that had been treated with 100 U/ml of IFN-g, and RT-PCR was performed as described in Materials and Methods. Amplified products for IL-6, IL-8, IL-15, MCP-1, and b-actin are shown. Product sizes of the genes for IL-6, IL-8, IL-15, and MCP-1 are 627, 292, 736, and 263 bp, respectively.

Article Snippet: Briefly, all sections were incubated with 2.5% normal horse serum for 30 min, followed by mAbs recognizing CD40 (Mab89, 10 mg/ ml, mouse IgG1; Coulter, Hialeah, FL), CD40L (thrombin receptor agonist peptide 1 (TRAP1), 10 mg/ml, mouse IgG1; Coulter), IL-15 (10 mg/ml, mouse IgG1; R&D Systems, Minneapolis, MN), and MCP-1 (10 mg/ml, mouse IgG1; Chemicon, Temecula, CA) or polyclonal Abs for IFN-g (5 mg/ml, rabbit IgG; Serotec, Oxford, U.K.) and IL-6 (5 mg/ml, goat IgG; Dako, Glostrup, Denmark) for 30 min.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Increased CD40 expression on muscle cells of polymyositis and dermatomyositis: role of CD40-CD40 ligand interaction in IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 production.

doi: 10.4049/jimmunol.164.12.6593

Figure Lengend Snippet: FIGURE 5. Expression of IL-6, IL-15, and MCP-1 in muscle tissue of PM/DM and normal controls. Some muscle cells were stained with anti- IL-6 mAb (A) or anti-MCP-1 mAb (C) in patient 2 (PM). These mAbs stained the cytoplasm of muscle cells. These IL-6- or MCP-1-positive mus- cle cells were smaller in diameter than muscle cells that were negative for stainings. Infiltrating MNCs also expressed MCP-1. Similar results were observed in patients 1, 6, and 7 (data not shown), but not in normal controls (F and H). The cytoplasm of muscle cells was strongly stained with anti- IL-15 mAb in patient 2 (B), although that of normal controls was margin- ally stained (G). Similar results were observed in patients 1, 6, and 7. D and E, Control staining of mouse IgG1 (D) and goat IgG (E) in patient 2. I and J, Control staining of mouse IgG1 (I) and goat IgG (J) in normal control. Original magnification, 3400

Article Snippet: Briefly, all sections were incubated with 2.5% normal horse serum for 30 min, followed by mAbs recognizing CD40 (Mab89, 10 mg/ ml, mouse IgG1; Coulter, Hialeah, FL), CD40L (thrombin receptor agonist peptide 1 (TRAP1), 10 mg/ml, mouse IgG1; Coulter), IL-15 (10 mg/ml, mouse IgG1; R&D Systems, Minneapolis, MN), and MCP-1 (10 mg/ml, mouse IgG1; Chemicon, Temecula, CA) or polyclonal Abs for IFN-g (5 mg/ml, rabbit IgG; Serotec, Oxford, U.K.) and IL-6 (5 mg/ml, goat IgG; Dako, Glostrup, Denmark) for 30 min.

Techniques: Expressing, Staining, Control

Journal: Journal of Immunology Research

Article Title: Flavokawain B Weakens Gastric Cancer Progression via the TGF- β 1/SMAD4 Pathway and Attenuates M2 Macrophage Polarization

doi: 10.1155/2022/4903333

Figure Lengend Snippet: Inactivation of the TSPAN12 expression and TGF- β 1/SMAD4 pathway in gastric cancer. (a–c) TSPAN12, TGF- β 1, and SMAD4 expression was tested in gastric cancer and normal tissues utilizing western blot. (d) Representative images of western blots. (e) Immunohistochemistry results of (a, b) TSPAN12, (c, d) TGF- β 1, and (e, f) SMAD4 in gastric cancer and normal tissues. ∗∗∗∗ p < 0.0001.

Article Snippet: Following antigen retrieval, the sections were blocked and stained with primary antibodies against TGF- β 1 (Abcam, USA, ab92486, 1 : 3000), SMAD4 (Abcam, ab40759, 1 : 3000), and TSPAN12 (Boster, A05472, 1 : 3000), followed by being incubated with secondary antibodies.

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: Journal of Immunology Research

Article Title: Flavokawain B Weakens Gastric Cancer Progression via the TGF- β 1/SMAD4 Pathway and Attenuates M2 Macrophage Polarization

doi: 10.1155/2022/4903333

Figure Lengend Snippet: FKB activates the TSPAN12 expression and TGF- β 1/SMAD4 pathway in gastric cancer cells. (a) SMAD4, (b) TGF- β 1, and (c) TSPAN12 expression was quantified in SGC-7901 cells treated with FKB. ∗ p < 0.05; ∗∗∗∗ p < 0.0001.

Article Snippet: Following antigen retrieval, the sections were blocked and stained with primary antibodies against TGF- β 1 (Abcam, USA, ab92486, 1 : 3000), SMAD4 (Abcam, ab40759, 1 : 3000), and TSPAN12 (Boster, A05472, 1 : 3000), followed by being incubated with secondary antibodies.

Techniques: Expressing

Journal: Journal of Immunology Research

Article Title: Flavokawain B Weakens Gastric Cancer Progression via the TGF- β 1/SMAD4 Pathway and Attenuates M2 Macrophage Polarization

doi: 10.1155/2022/4903333

Figure Lengend Snippet: FKB activates TSPAN12 expression and TGF- β 1/SMAD4 pathway in vivo. (a–c) Immunohistochemistry results of TGF- β 1, SMAD4, and TSPAN12 proteins in gastric cancer tissues of FKB treatment and control groups. Bar = 20 μ m. (d) Representative images of western blot. (e–g) TGF- β 1, SMAD4, and TSPAN12 expression was examined in gastric cancer tissues of nude mice between FKB treatment and control groups using western blot. (h) FKB treatment prolonged the survival time of nude mice xenografted with SGC-7901 cells. (i) FKB treatment lowered the tumor weight of nude mice xenografted with SGC-7901 cells. ∗∗∗∗ p < 0.0001.

Article Snippet: Following antigen retrieval, the sections were blocked and stained with primary antibodies against TGF- β 1 (Abcam, USA, ab92486, 1 : 3000), SMAD4 (Abcam, ab40759, 1 : 3000), and TSPAN12 (Boster, A05472, 1 : 3000), followed by being incubated with secondary antibodies.

Techniques: Expressing, In Vivo, Immunohistochemistry, Control, Western Blot