mouse anti-human adiponectin  (GeneTex)

Journal: Scientific reports

Article Title: Regulation and function of adiponectin in the intestinal epithelial cells in response to Trichinella spiralis infection.

doi: 10.1038/s41598-023-41377-x

Figure Lengend Snippet: Figure 1. The induction of adiponectin in small intestinal tissue during T. spiralis infection. T. spiralis larvae were orally gavaged into BALB/c mice. Blood and small intestinal tissue were harvested at 7 and 14 days after infection to evaluate adiponectin and leptin expression using ELISA analysis and quantitative real-time PCR. (A) ELISA analysis of adiponectin, leptin, and the adiponectin/leptin ratio in serum from uninfected mice or mice infected with T. spiralis. (B) Quantitative real-time PCR analysis of adiponectin and leptin and the representative data of worm number in the intestinal tissue from uninfected mice or mice infected with T. spiralis. The mRNA expression data are presented as fold induction over actin (Actb) expression, with the mRNA levels in uninfected mice set as 1. (C) ELISA analysis of adiponectin and leptin in jejunum lysate from uninfected mice or mice infected with T. spiralis. (D,E) The correlation between intestinal worm number and the mRNA and protein expression levels of adiponectin (D) and leptin (E) in the intestinal tissue from mice infected with T. spiralis for 7 days. Graphs depict individual mice and mean ± SD is representative of three pooled independent experiments, with n = 6 mice per group. Significance was determined using one-way ANOVA followed by Turkey post hoc analysis and Spearman’s rank test. Correlation coefficients (r) and p values are provided. (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Anti-mouse adiponectin antibody (10 μg/mL, AF119; R&D system) was used for neutralization of adiponectin.

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Scientific reports

Article Title: Regulation and function of adiponectin in the intestinal epithelial cells in response to Trichinella spiralis infection.

doi: 10.1038/s41598-023-41377-x

Figure Lengend Snippet: Figure 2. The induction of adiponectin is associated with epithelial cell-derived cytokines in intestinal tissue upon T. spiralis infection. (A–C) T. spiralis larvae were orally gavaged into BALB/c mice. Jejunum tissue was collected at 7 days after infection to evaluate the mRNA expression of adiponectin, epithelial cell-derived cytokines, type 2 cytokine, proinflammatory, and regulatory cytokines using quantitative real-time PCR. The correlation between gene expression level of adiponectin and (A) epithelial cell-derived cytokines (Il25, Il33, Tslp), (B) type 2 cytokine (Il4, Il5, Il13) and (C) proinflammatory cytokine (Il17, Tnfα, Ifnγ) or regulatory cytokine (Il10) in jejunum tissue of mice infected with T. spiralis, with n = 12 mice was analyzed using Spearman’s rank test. Correlation coefficients (r) and p values are provided. (*p < 0.05, **p < 0.01).

Article Snippet: Anti-mouse adiponectin antibody (10 μg/mL, AF119; R&D system) was used for neutralization of adiponectin.

Techniques: Derivative Assay, Infection, Expressing, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Scientific reports

Article Title: Regulation and function of adiponectin in the intestinal epithelial cells in response to Trichinella spiralis infection.

doi: 10.1038/s41598-023-41377-x

Figure Lengend Snippet: Figure 3. IL-25 signaling is required to induce adiponectin expression in the intestine during T. spiralis infection. (A,B) Wild-type and Il17rb−/− mice were infected with T. spiralis larvae by oral gavage. Jejunum tissue was collected at 7 days after infection to evaluate the expression of adiponectin and leptin using quantitative real-time PCR and ELISA analysis. (A) The mRNA expression of adiponectin and leptin in the jejunum tissue from uninfected or T. spiralis-infected wild-type and Il17rb−/− mice. Data are presented as fold induction over actin (Actb) expression, with the mRNA expression levels in uninfected wild-type mice set as 1. (B) ELISA analysis of adiponectin and leptin in jejunum tissue lysate from uninfected or T. spiralis-infected wild-type and Il17rb−/− mice. Graphs depict individual mice and mean ± SD is representative of three pool independent experiments, with n = 6 mice per group. Significance was determined using two-way ANOVA followed by Bonferroni’s post-test analysis (**p < 0.01, ***p < 0.001). (C) Wild-type and iIL-25Tg mice were orally gavaged with 400 larvae of T. spiralis. At 7 days of infection, mice were euthanized and jejunum tissue was collected for measurement of adiponectin and leptin expression. (left) Quantitative real-time PCR analysis of adiponectin, Data are presented as fold induction over actin (Actb) expression, with the mRNA expression levels in uninfected wild-type mice set as 1 and (right) ELISA analysis of adiponectin in jejunum tissue lysate from uninfected or T. spiralis-infected wild-type and iIL-25Tg mice. Graphs depict individual mice and mean ± SD is representative from three pooled independent experiments, with n = 6 mice per group. Significance was determined using one-way ANOVA followed by Turkey post hoc analysis (*p < 0.05, ***p < 0.001).

Article Snippet: Anti-mouse adiponectin antibody (10 μg/mL, AF119; R&D system) was used for neutralization of adiponectin.

Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Scientific reports

Article Title: Regulation and function of adiponectin in the intestinal epithelial cells in response to Trichinella spiralis infection.

doi: 10.1038/s41598-023-41377-x

Figure Lengend Snippet: Figure 4. Adiponectin is mainly expressed by intestinal epithelial cells during T. spiralis infection. (A,B) BALB/c mice were orally gavaged with 400 larvae of T. spiralis. At 7 days post-infection, mice were sacrificed and the small intestines were harvested. Intestinal epithelial cells (IEC) or lamina propria cells (LPL) were isolated for the assessment of adiponectin and IL-25 expression using quantitative real-time PCR and western analysis. (A) Quantitative real-time PCR analysis of Adipoq and Il25 mRNA expression in isolated intestinal epithelial cells and lamina propria cells from uninfected and T. spiralis-infected mice. Data are presented as fold induction over actin (Actb) expression, with the mRNA expression levels from naive mice set as 1. (B) Western blot analysis of adiponectin and β-actin in isolated intestinal epithelial cells from uninfected and T. spiralis- infected mice. Lane M represents molecular weight protein marker (kDa). The molecular weights of adiponectin and β-actin are 27 kDa and 42 kDa, respectively. Bar graph represents the protein level by measuring intensity of protein bands in arbitrary units. the intensity of β-actin was normalized with the intensity of adiponectin. The full-length blots are presented in Supplementary Fig. S1. (C) Wild-type and Il17rb−/− mice were infected with T. spiralis for 7 days and the intestinal epithelial cells were isolated and analyzed for the quantification of adiponectin and IL-25 expression using quantitative real-time PCR. Data are presented as fold induction over actin (Actb) expression, with the mRNA levels in intestinal epithelial cells from naïve wild-type mice set as 1. Graphs depict mean ± SD of at least three independent experiments, with n = 3 mice per group. Significance was determined using two-way ANOVA followed by Bonferroni’s post-test analysis and Student’s t-test analysis (**p < 0.01, ***p < 0.001).

Article Snippet: Anti-mouse adiponectin antibody (10 μg/mL, AF119; R&D system) was used for neutralization of adiponectin.

Techniques: Infection, Isolation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Molecular Weight, Marker

Journal: Scientific reports

Article Title: Regulation and function of adiponectin in the intestinal epithelial cells in response to Trichinella spiralis infection.

doi: 10.1038/s41598-023-41377-x

Figure Lengend Snippet: Figure 5. IL-25-induced adiponectin expression promotes the upregulation of occludin and CCL17 in isolated intestinal epithelial cells. Isolated intestinal epithelial cells from wild-type and Il17rb−/− mice were treated in the presence or absence of IL-25 for 36 h and analyzed for the expression of indicated genes using quantitative real-time PCR. (A) Quantitative real-time PCR analysis of adiponectin (Adipoq), tight junction-associated genes (Ocln, Cldn1, Jam1), mucus-associated genes (Clca1), and chemokines (Ccl11, Ccl17, Ccl24). Data are presented as fold induction over actin (Actb) expression, with the mRNA levels in the control untreated cells set as 1. (B) The isolated intestinal epithelial cells from wild-type mice were treated with IL-25 (100 ng/mL) in the presence of adiponectin neutralizing antibody (anti-ADPN, 10 μg/mL) or isotype control (10 μg/mL) for 36 h. and analyzed for the expression of Adipoq, Ocln, Ccl17, and Il25 using quantitative real-time PCR. Data are presented as fold induction over actin (Actb) expression, with the mRNA levels in the control untreated cell set as 1. Graphs depict mean ± SD of three independent experiments, with n = 3 mice per group. Significance was determined using two-way ANOVA followed by Bonferroni’s post-test analysis and one-way ANOVA followed by Turkey post hoc analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Anti-mouse adiponectin antibody (10 μg/mL, AF119; R&D system) was used for neutralization of adiponectin.

Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Control

Journal: Scientific reports

Article Title: Regulation and function of adiponectin in the intestinal epithelial cells in response to Trichinella spiralis infection.

doi: 10.1038/s41598-023-41377-x

Figure Lengend Snippet: Figure 6. Adiponectin attenuates LPS-induced barrier dysfunction and prevents larval invasion in Caco2 cell monolayers. (A–C) Caco2 cells were grown in transwell insert of 24-well plate for 21 days. Cells were pre-treated with 1 µg/mL human recombinant adiponectin for 24 h. DMEM with 0.1% FBS was used as vehicle control. Pre- treated cells were then stimulated with 0.1 and 1 µg/mL LPS for 24 h before analysis of barrier integrity and gene expression. (A) Transwell system used for investigating the effect of adiponectin in controlling LPS‑induced barrier dysfunction Caco-2 cells. (B) (%) TEER values in each condition treatment. (C) Quantitative real- time PCR analysis of tight junction-associated genes (Ocln, Cldn1, Zo1), antimicrobial peptides (Defa5), and proinflammatory cytokine (Tnfα) mRNA expression. Data are presented as fold induction over actin (Actb) expression, with the mRNA levels in the control untreated cell set as 1. (D,E) Caco2 cells were grown in 24-well plate for 21 days. Cells were pre-treated with 1 µg/mL human recombinant adiponectin for 24 h. DMEM with 0.1% FBS was used as vehicle control. In vitro larval invasion assay was performed for 2 h. The PMSF-pretreated ILLs were used as an inhibitor of larval invasion. The pictures of larval invasion were captured under an inverted microscope (Scale bar 500 μm) and the number of invaded larvae and non-invaded larvae were counted under an inverted light microscope. (D) Representative data of invaded larvae (migratory path showed as black arrows, Invaded larvae penetrate in cell monolayers show as red arrow) and non-invaded larvae (their coiled on the cell monolayer without movement show as yellow arrow) at 2 h after infection from different treatment groups, including non-infection, Caco2 cells pre-treated with vehicle control, Caco2 cells pre-treated with adiponectin, and Caco2 cells with ILLs that pre-treated with PMSF. (E) (%) Larva invasion (above) and (%) Larva invasion inhibition (below) from different treatment groups. Graphs depict mean ± SD of three independent experiments. Significance was determined using one-way ANOVA followed by Turkey post hoc analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Anti-mouse adiponectin antibody (10 μg/mL, AF119; R&D system) was used for neutralization of adiponectin.

Techniques: Recombinant, Control, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, In Vitro, Invasion Assay, Inverted Microscopy, Light Microscopy, Infection, Inhibition

Journal: Scientific reports

Article Title: Regulation and function of adiponectin in the intestinal epithelial cells in response to Trichinella spiralis infection.

doi: 10.1038/s41598-023-41377-x

Figure Lengend Snippet: Figure 7. Administration of adiponectin induced intestinal IL-13 secretion and promoted worm expulsion. BALB/c mice were orally gavaged with 400 larvae of T. spiralis and given intraperitoneally with recombinant adiponectin (20 µg/mouse) daily for six consecutive days. PBS sterile (pH 7.2–7.4) was injected as vehicle control. At 7 days after infection, mice were sacrificed. (A) The whole intestines were collected and subjected for worm burden analysis. (B) Jejunum tissue lysate of uninfected or T. spiralis-infected mice treated with recombinant adiponectin or PBS at 7 days post-infection was measured for the secretion levels of IL-4, IL-13, IFNγ and IL-17 using ELISA analysis. Graphs depict mean ± SD is representative from two pooled independent experiments, with n = 4 mice per group. Significance was determined using Student’s t-test analysis and one-way ANOVA followed by Turkey post hoc analysis (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Anti-mouse adiponectin antibody (10 μg/mL, AF119; R&D system) was used for neutralization of adiponectin.

Techniques: Recombinant, Sterility, Injection, Control, Infection, Enzyme-linked Immunosorbent Assay

Journal: Archives of Gynecology and Obstetrics

Article Title: Glycosylated fibronectin as a first trimester marker for gestational diabetes

doi: 10.1007/s00404-020-05670-8

Figure Lengend Snippet: a Glycosylated fibronectin concentrations in BMI subgroups of 20–25 and 30–35 kg/m 2 in the study and the control group. b Effect of smoking on concentrations of glycosylated fibronectin in the control and GDM groups. c Multiple of median (MoM) values for fibronectin and glycosylated fibronectin between control and GDM groups. d Multiple of median (MoM) values for adiponectin and glycosylated adiponectin between control and GDM groups

Article Snippet: Standard adiponectin assay was done by coating 96-well plates with mouse monoclonal IgG antibody against human adiponectin (MAB10651, R&D Systems, Abingdon, UK).

Techniques: Control

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: Relationship between adiponectin expression ratio and demographic characteristics of non-small cell lung cancer (NSCLC) patients.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: Survival curve of non-small cell lung cancer patients with high and low expression of adiponectin assessed using Kaplan–Meier analysis.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: Multivariate cox regression analysis of mortality.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Expressing

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) Samples (lung cancer and the corresponding normal adjacent lung tissues) were analyzed with antibodies to adiponectin, adiponectin receptor 1, and adiponectin receptor 2 by immunohistochemical staining (DAB staining and hematoxylin counterstaining). For negative controls, the antibody was replaced with control IgG. (B-C) Expression levels of adiponectin, adiponectin receptor 1, and adiponectin receptor 2 in three pairs of lung cancer and normal tissues analyzed by western blotting. (D–F) Gelatin zymography was used to analyze the activities of MMP-2/MMP-9, MMP-1/MMP-3, and MMP-13/MMP-14. * p < 0.05 vs. normal lung tissue, with representative data from three different patients with NSCLC. T, tumor; N, normal; ADN, Adiponectin; AdipoR1, adiponectin receptor 1; AdipoR2, adiponectin receptor 2.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Immunohistochemical staining, Staining, Control, Expressing, Western Blot, Zymography

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) Cell viability was analyzed by the MTT assay. The cytotoxic effect of curcumin was obvious at concentrations >75 μM. (B) Expression levels of adiponectin, adiponectin receptor 1, and adiponectin receptor 2 in A549 cells were analyzed by western blotting at different curcumin concentrations. (C–D) Exogenous induction and silencing of adiponectin expression in A549 cells. Adiponectin mRNA/protein expression in A549 cells increased significantly after exogenous adiponectin expression as analyzed by real-time PCR. Adiponectin expression decreased significantly after siRNA-mediated adiponectin silencing. NC-siRNA served as a negative control. Representative photos of three independent experiments. * p < 0.05 vs. control. The assays were conducted in triplicate.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: MTT Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Negative Control, Control

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) Cellular migration was determined using scratch wound assay and analyzed using the Wimasis WimScratch software. The migratory ability of A549 cells was significantly inhibited by increasing curcumin dosage compared with the control group ( p < 0.05). (B) Invasiveness was determined by Matrigel-coated Boyden chamber assay. Curcumin significantly decreased the invasive ability of A549 cells ( p < 0.05) when the curcumin concentration was >25 uM. (C–D) The application of recombinant adiponectin (rADN) significantly increased the migratory and invasive ability of A549 cells ( p < 0.05). The results of three independent experiments are shown; the assays were conducted in triplicate.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Migration, Scratch Wound Assay Assay, Software, Control, Boyden Chamber Assay, Concentration Assay, Recombinant

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A–C) The expression levels of adiponectin, adiponectin receptor 1, and adiponectin receptor 2 were analyzed by western blotting after transfection with adiponectin vector or silencing by siADN, siAdipoR1, and siAdipoR2. (D–E) The migratory and invasive ability of A549 cells was inhibited after silencing of the adiponectin receptor 1 expression by transfection with siAdipoR1.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) A549 cells were treated with various PI3K/AKT and MAP kinase pathway inhibitors {(PI3K (LY294002; 10 μM), AKT (API-59; 3 μM), MAPK inhibitors [PD98059 (10 μM), SB203580 (10 μM), and SP600125 (10 μM) for ERK, p38–MAPK, and JNK, respectively]} for 1 h and later with curcumin (50 μM) for 24 h. Adiponectin expression was analyzed by western blotting. (B) AKT expression was analyzed by western blotting with different concentrations of recombination adiponectin. (C) The activity of p65/p50 of A549 cells was analyzed by EMSA after treatment with recombinant adiponectin or AKT inhibitor (API-59; 3 μM), respectively.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Expressing, Western Blot, Activity Assay, Recombinant

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) A549 cells treated with curcumin, silenced, or transfected with adiponectin were lysed and adiponectin was co-immunoprecipitated using adiponectin antibody. Immunoblotting with the indicated antibodies confirmed the co-precipitation of adiponectin monomer, dimer, and multimer. (B) Co-immunoprecipitation was performed using anti-adiponectin and anti-p65 antibodies. The p65 elute was separated on SDS-PAGE and immunoblotted with the corresponding antibodies, which showed substantial association of adiponectin with the p65 component. (C) Activation of NF-κB by overexpression or silencing of adiponectin was determined by EMSA. Adiponectin increased the nuclear translocation of p65/p50 and NF-κB activation, but silencing adiponectin expression had the opposite effect.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Transfection, Immunoprecipitation, Western Blot, SDS Page, Activation Assay, Over Expression, Translocation Assay, Expressing

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: Gelatin zymography was used to analyze d the activities of MMP-2/MMP-9, MMP-1/MMP-3, and MMP-13/MMP-14. (A–F) The activities of MMP-9, -1, and -14 were increased after the overexpression of adiponectin and decreased with the silencing of adiponectin. Expression of MMP-3 and -13 did not change regardless of adiponectin augmentation or silencing.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Zymography, Over Expression, Expressing

Journal: PLoS ONE

Article Title: Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

doi: 10.1371/journal.pone.0144462

Figure Lengend Snippet: (A) The tumor sizes of curcumin-treated mice were decreased significantly compared to those of the control group after 14 days. (B) Tumor adiponectin expression of curcumin-treated mice was decreased significantly compared to that of the control group. (C) Expression of both adiponectin receptor 1 and receptor 2 in the curcumin-treated mice did not change in vivo . (D–F) Expression levels of MMP-2, -9, -3, -13, and -14 were decreased with curcumin treatment in vivo . MMP-1 expression was not altered.

Article Snippet: Western blotting was performed as described previously [ ], after which the membranes were treated with PBS containing 0.05% Tween 20 and 2% skimmed milk for 1 h at room temperature and incubated separately with mouse anti-human adiponectin (GeneTex, Irvine, CA, USA), AdipoR1, AdipoR2, p65, p50, MMPs (2, 9, 1, 3, 13, 14) (Santa Cruz, CA, USA), Histone H1, AKT/pAKT, P38/pP38 (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA) for 1 h. After washing, the membranes were incubated with horseradish peroxidase–conjugated rabbit anti-goat or mouse IgG at room temperature.

Techniques: Control, Expressing, In Vivo