Journal: International Journal of Molecular Medicine

Article Title: Margatoxin mitigates CCl4-induced hepatic fibrosis in mice via macrophage polarization, cytokine secretion and STAT signaling

doi: 10.3892/ijmm.2019.4395

Figure Lengend Snippet: MgTX regulates cytokine secretion in vivo . A total of 3 different MgTX concentrations were injected into mice via intraperitoneal injection. The cytokines in mouse serum were measured using ELISA kits, MgTX(H):0.41 mg/Kg, MgTX(M):0.14 mg/Kg, MgTX(L):0.04 mg/Kg. (A-F) ELISAs to detect the levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-20) in-mouse serum. Cytokine levels of IL-10 were inhibited compared with the HF model group, whereas these levels were increased compared with the HF model group treated with MgTX. The cytokine levels of TGF-β group (n=6) increased in the model group, while there was no significant difference observed in the HF model group treated with MgTX. * P<0.05 vs. control group; # P<0.05 vs. model group. n=6. MgTX, margatoxin; TNF-α, tumor necrosis factor-α; IL, interleukin; TGF-β, transforming growth factor-β; HF, hepatic fibrosis.

Article Snippet: Primary antibodies against δ-catenin (1:300, cat. no. bs-7000R, Bioss), α-SMA (1:300, cat. no. bs-0189R, Bioss), collagen I (1:300, cat. no. bs-10423R, Bioss), MMP12 (1:600, cat. no. 22989-1-AP, Proteintech), MMP13 (1:300, cat. no. bs-10581R, Bioss), POSTN (1:300, cat. no. bs-4994R, Bioss), CCL2 (1:500, cat. no. bs1101R, Bioss), Kv1.3 (1:300, cat. no. bs-10229R, Bioss), iNOS (1:1,000, cat. no. ab4999, Abcam), TNF-α (1:500, cat. no. BS1857, Bioworld), IL-β (1:300, cat. no. bs0812R, Bioss), IL-10 (1:1,000, cat. no. bs0698R, Bioss), CD163 (1:300, cat. no. bs2527R, Bioss), Arg-1 (1:200, cat. no. sc-47715, Santa Cruz Biotechnology), Mrc2 (1:500, cat. no. ab70132, Abcam) and β-actin (1:500, cat. no. TA-09, Zs-BIO) were used.

Techniques: In Vivo, Injection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Medicine

Article Title: Margatoxin mitigates CCl4-induced hepatic fibrosis in mice via macrophage polarization, cytokine secretion and STAT signaling

doi: 10.3892/ijmm.2019.4395

Figure Lengend Snippet: MgTX regulates macrophage polarization in vitro . Reverse transcription-quantitative PCR and western blot analysis were performed to detect the expression of M1 markers and M2 markers in RAW264.7 cells. (A-H) mRNA expression levels of iNOS, CCL2, TNF-α and IL-1β were downregulated by MgTX in M1 phenotype macrophages. mRNA expression levels of IL-10, CD163, Arg-1, MAC-2 were upregulated in M2 phenotype macrophages treated with MgTX compared with M2 phenotype macrophages. (I-Q) protein expression levels of M1 markers (iNOS, CCL2, TNF-α and IL-1β) and protein expression levels of M2 markers (IL-10, CD163, Arg-1, Mrc2) were measured by western blot analysis. * P<0.05, ** P<0.01, M1 vs. M1 + MgTX group, M2 vs. M2 + MgTX group. control group (N), n=3. MgTX, margatoxin; CCL2, C-C motif chemokine ligand 2; TNF-α, tumor necrosis factor-α; IL, interleukin; CD, cluster of differentiation.

Article Snippet: Primary antibodies against δ-catenin (1:300, cat. no. bs-7000R, Bioss), α-SMA (1:300, cat. no. bs-0189R, Bioss), collagen I (1:300, cat. no. bs-10423R, Bioss), MMP12 (1:600, cat. no. 22989-1-AP, Proteintech), MMP13 (1:300, cat. no. bs-10581R, Bioss), POSTN (1:300, cat. no. bs-4994R, Bioss), CCL2 (1:500, cat. no. bs1101R, Bioss), Kv1.3 (1:300, cat. no. bs-10229R, Bioss), iNOS (1:1,000, cat. no. ab4999, Abcam), TNF-α (1:500, cat. no. BS1857, Bioworld), IL-β (1:300, cat. no. bs0812R, Bioss), IL-10 (1:1,000, cat. no. bs0698R, Bioss), CD163 (1:300, cat. no. bs2527R, Bioss), Arg-1 (1:200, cat. no. sc-47715, Santa Cruz Biotechnology), Mrc2 (1:500, cat. no. ab70132, Abcam) and β-actin (1:500, cat. no. TA-09, Zs-BIO) were used.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: Cells

Article Title: Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins

doi: 10.3390/cells10020274

Figure Lengend Snippet: Galectins induce CXCL10 in a cell type-specific manner. ( A ) SDS-polyacrylamide gel electrophoresis of recombinant human galectins (rhGal) stained with Coomassie Blue. Molecular weights of protein standards are indicated on the left. The hemagglutination assay was performed using serial two-fold dilutions of the recombinant protein. The bottom row contains protein incubated with β-lactose, a competitive carbohydrate inhibitor of galectin binding. ( B ) Cultures of human corneal fibroblasts, human corneal epithelial cells, THP-1 monocytes and human umbilical vein endothelial cells (HUVECs) were incubated with 50 µg/mL rhGal-1, -3 and -8 or 50 µg/mL IFN-γ. Quantitative real-time PCR was used to determine the levels of CXCL10 mRNA after 6 h of incubation ( n = 3 independent experiments), whereas ELISA was used to determine the levels of CXCL10 protein in cell culture supernatants after 24 h of incubation ( n = 6 independent experiments). The data represent the mean ± SEM.

Article Snippet: A mouse monoclonal antibody against CXCL10 (1.2 μg/mL; MAB266, R&D Systems) was added to the lower compartment of the chamber in neutralization studies.

Techniques: Polyacrylamide Gel Electrophoresis, Recombinant, Staining, Hemagglutination Assay, Incubation, Binding Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cells

Article Title: Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins

doi: 10.3390/cells10020274

Figure Lengend Snippet: Galectins differentially dictate fibroblast-dependent immune cell chemotaxis. ( A ) Recombinant human galectin (rhGal)-1, -3 and -8 were incubated with fibroblasts for 24 h. The conditioned media were pre-cleared with β-lactose Sepharose beads three times to remove galectins prior to chemotaxis assays. The presence of galectins in pre-cleared conditioned media was evaluated by immunoblotting. ( B ) Quantification by flow cytometry of the migration of CFSE-labeled THP-1 and Jurkat cells to conditioned media from fibroblasts treated for 24 h with increasing concentrations of recombinant galectins or BSA ( n = 3 independent experiments performed in duplicate). ( C ) Migration of CFSE-labeled THP-1 and Jurkat cells to conditioned media from fibroblasts treated for 24 h with 50 μg/mL of recombinant galectins or BSA. A CXCL10 neutralizing antibody was added to the conditioned media in neutralization studies ( n = 4–5 independent experiments in duplicate). The data in ( B ) represent the mean ± SEM. The box and whisker plots show the 25 and 75 percentiles (box), the median, and the minimum and maximum data values (whiskers). Significance was determined using Student’s t test. ** p < 0.01.

Article Snippet: A mouse monoclonal antibody against CXCL10 (1.2 μg/mL; MAB266, R&D Systems) was added to the lower compartment of the chamber in neutralization studies.

Techniques: Chemotaxis Assay, Recombinant, Incubation, Western Blot, Flow Cytometry, Migration, Labeling, Neutralization, Whisker Assay