Journal: Haematologica

Article Title: Resistin induces multidrug resistance in myeloma by inhibiting cell death and upregulating ABC transporter expression

doi: 10.3324/haematol.2016.154062

Figure Lengend Snippet: Resistin protects myeloma cells from chemotherapy-induced apoptosis. (A) Human myeloma cell lines ARP-1, MM.1S, and U266 were cultured in medium containing melphalan (25 μM) plus resistin (0, 10, 25, 50, 100, or 200 ng/mL) for 24 h; cells without melphalan treatment served as a control. Apoptosis in the cultured cells was determined by using an annexin V binding assay. The percentages of apoptotic cells in each of the three cell lines are shown. (B, C, D) ARP-1, MM.1S, and RPMI8226 cells were cultured in medium containing melphalan (Mel; 25 μM), bortezomib (BTZ; 5 nM), or carfilzomib (CFZ; 20 nM) with or without resistin (50 ng/mL) for 24 h. Cells cultured without the chemotherapy agents or resistin served as controls. Percentages of apoptotic cells are shown. (E) CD138 + plasma cells were isolated from bone marrow aspirates of five patients with multiple myeloma and cultured with melphalan (25 μM) without or with resistin (50 ng/mL) for 24 h. Percentages of apoptotic myeloma cells are shown. Results shown represent three to five independent experiments. * P <0.05; ** P <0.01.

Article Snippet: Recombinant human resistin was purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: Cell Culture, Control, Binding Assay, Clinical Proteomics, Isolation

Journal: Haematologica

Article Title: Resistin induces multidrug resistance in myeloma by inhibiting cell death and upregulating ABC transporter expression

doi: 10.3324/haematol.2016.154062

Figure Lengend Snippet: Resistin activates anti-apoptotic signaling pathways in myeloma cells. (A, B) ARP-1 and MM.1S myeloma cells were treated with melphalan (Mel; 25 μM) and/or resistin (50 ng/mL) for 24 h. Western blot analysis shows (A) the levels of cleaved (c) caspase (Cas)-9, Cas-3, and PARP, and (B) the expression of the mitochondria-related anti-apoptotic proteins Bcl-2 and Bcl-xL and the pro-apoptotic protein Bax in the cells. Cells cultured without treatment served as controls. (C) ARP-1 and MM.1S cells were cultured in medium with or without resistin (0, 50 ng/mL, or 100 ng/mL) for 12 h. Western blot analysis shows the levels of non-phosphorylated and phosphorylated (p) IκBα, Akt, and ERK1/2 in the cells treated with resistin. GAPDH served as a protein loading control. (D–F) ARP-1 or MM.1S cells were pretreated with (D) 1 μM NF-κB inhibitor Ro106, (E) 0.5 μM PI3K inhibitor LY294002, or (F) 1 μM MEK1/2 inhibitor U0126 for 1 h, followed by treatment with melphalan (25 μM) and/or resistin (50 ng/mL) for 24 h. The annexin V binding assay shows the percentages of apoptotic cells for each treatment condition. Cells cultured with none of the inhibitors served as controls. Results shown represent three independent experiments. * P <0.05.

Article Snippet: Recombinant human resistin was purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: Protein-Protein interactions, Western Blot, Expressing, Cell Culture, Control, Binding Assay

Journal: Haematologica

Article Title: Resistin induces multidrug resistance in myeloma by inhibiting cell death and upregulating ABC transporter expression

doi: 10.3324/haematol.2016.154062

Figure Lengend Snippet: Resistin increases the expression of ABC transporters in myeloma cells. ARP-1 and MM.1S myeloma cells were cultured with resistin (50 ng/mL) for 12 h. Some of the cultured cells were labeled with eFluxx-ID gold fluorescent dye and further analyzed by flow cytometry. Others were subjected to RNA or protein extraction for real-time PCR or western blot analysis. (A) Intracellular eFluxx-ID gold fluorescence intensity was quantified. PBS, phosphate-buffered saline solution (controls). (B) Real-time PCR shows relative mRNA expression of ABC transporter genes. (C) Western blot analysis shows expression of ABCG2 and ABCC5 proteins. Cells cultured without resistin served as controls. GAPDH served as a protein loading control. (D) Real-time PCR analysis shows relative expression levels of ABCC5 and ABCG2 mRNA in ARP-1 or MM.1S cells bearing non-targeted siRNA ( si Ctrl) or the pooled siRNA of ABCC5 ( si C5) and ABCG2 ( si G2). (E) The percentages of apoptotic cells in si Ctrl- or both si C5- and si G2-expressing ARP-1 or MM.1S cells treated with or without resistin or melphalan (Mel) are shown. Results are representative of three independent experiments. * P <0.05; ** P <0.01.

Article Snippet: Recombinant human resistin was purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: Expressing, Cell Culture, Labeling, Flow Cytometry, Protein Extraction, Real-time Polymerase Chain Reaction, Western Blot, Fluorescence, Saline, Control

Journal: Haematologica

Article Title: Resistin induces multidrug resistance in myeloma by inhibiting cell death and upregulating ABC transporter expression

doi: 10.3324/haematol.2016.154062

Figure Lengend Snippet: Resistin reduces the methylation of ABCG2 and ABCC5 gene promoters. ARP-1 and MM.1S myeloma cells were cultured with resistin (50 ng/mL) for 12 h. (A, B) Genomic DNA was extracted from cultured cells for methylation-specific PCR analysis. (A) Representative images of the methylated (M) and un-methylated (U) CpG sites and (B) quantitative data of M/U ratios in the promoters of ABCG2 or ABCC5 genes. PBS, phosphate-buffered saline solution (controls). (C, D) Total RNA and total proteins were extracted from cultured cells for real-time reverse transcriptase-PCR or western blot analysis. (C) Relative DNMT1 , DNMT3a , and DNMT3b mRNA expression and (D) DNMT1 and DNMT3a protein expression. Cells cultured without resistin served as controls. GAPDH served as a protein loading control in western blot analysis. Results shown are representative of three independent experiments. * P <0.05; ** P <0.01.

Article Snippet: Recombinant human resistin was purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: Methylation, Cell Culture, Saline, Reverse Transcription, Western Blot, Expressing, Control

Journal: Haematologica

Article Title: Resistin induces multidrug resistance in myeloma by inhibiting cell death and upregulating ABC transporter expression

doi: 10.3324/haematol.2016.154062

Figure Lengend Snippet: Resistin protects myeloma from chemotherapy in vivo . SCID mice were injected with ARP-1 myeloma cells (5×10 5 cells per mouse) directly into the femur (n=5 mice per group). Three weeks after ARP-1 cell injection, mice began intraperitoneal treatment with melphalan (Mel; 50 μg/mouse), resistin (20 μg/mouse), or both every 3 days for 3 weeks. After treatment, the mouse sera were subjected to enzyme-linked immusorbent assay to measure M-protein levels. After the mice had been euthanized, the cells flushed from each mouse’s femoral bone marrow cavity were labeled with an antibody against human CD138, and the CD138 + cells were sorted by flow cytometry. CD138 + cells were subjected to an annexin V binding assay to determine cell apoptosis. The mouse femora were analyzed with an in situ TUNEL assay. Mice that received neither melphalan nor resistin served as controls. (A) Relative levels of M-proteins. (B) Percentages of CD138 + cells. (C) Percentages of apoptotic CD138 + cells. (D) Representative images of TUNEL + cells in bone marrow. (E) Quantitative analysis of TUNEL staining. Bar: 20 μm. Original magnification × 200. The results shown represent averages ± SD (n = 5 mice/group, 3 replicate studies). * P <0.05.

Article Snippet: Recombinant human resistin was purchased from PeproTech (Rocky Hill, NJ, USA).

Techniques: In Vivo, Injection, Labeling, Flow Cytometry, Binding Assay, In Situ, TUNEL Assay, Staining

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Effects of resistin on porcine ovarian follicle steroidogenesis in prepubertal animals: an in vitro study

doi: 10.1186/1477-7827-11-45

Figure Lengend Snippet: Effects of recombinant human resistin (0.1, 1, 10, and 100 ng/ml) on steroid hormone (i.e., progesterone [P4], androstendione [A4], testosterone [T], and estradiol [E2]) secretion in culture medium from A) small, B) medium and C) large ovarian follicles. ELISA experiments were performed three independent times. In each experiment six ovaries from three different animals were selected. Data were plotted as mean±S.E.M. Different letters indicate statistically significant differences among groups ( P < 0.05).

Article Snippet: Recombinant human resistin was obtained from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Effects of resistin on porcine ovarian follicle steroidogenesis in prepubertal animals: an in vitro study

doi: 10.1186/1477-7827-11-45

Figure Lengend Snippet: Effects of recombinant human resistin (0.1, 1, 10, and 100 ng/ml) on the mRNA expression of steroidogenic enzymes (i.e., CYP11A1, 3βHSD, CYP17A1, 17βHSD, and CYP19A1 ) in A) small, B) medium and C) large ovarian follicles. The expression of mRNA has been determined by real time-PCR. The expression of each gene was normalized to the expression of GAPDH. Real time PCR experiments were performed three independent times. In each experiment six ovaries from three different animals were selected. Data were plotted as mean±S.E.M. Statistical significance is indicated by * P < 0.05.

Article Snippet: Recombinant human resistin was obtained from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA).

Techniques: Recombinant, Expressing, Real-time Polymerase Chain Reaction

Journal: Reproductive Biology and Endocrinology : RB&E

Article Title: Effects of resistin on porcine ovarian follicle steroidogenesis in prepubertal animals: an in vitro study

doi: 10.1186/1477-7827-11-45

Figure Lengend Snippet: Effects of recombinant human resistin. (0.1, 1, 10, and 100 ng/ml) on the protein expression of steroidogenic enzymes CYP11A1, 3βHSD, CYP17A1, 17βHSD, and CYP19 small, medium and large ovarian follicles. The representative samples of western blots are shown in the panels. Western blot experiments were repeated three independent times using samples from three different animals. Data were plotted as mean±S.E.M. Statistical significance is indicated by * P < 0.05.

Article Snippet: Recombinant human resistin was obtained from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA).

Techniques: Recombinant, Expressing, Western Blot

Journal: Cancer Science

Article Title: Resistin facilitates metastasis of lung adenocarcinoma through the TLR 4/Src/ EGFR / PI 3K/ NF ‐κB pathway

doi: 10.1111/cas.13704

Figure Lengend Snippet: Human resistin is upregulated in lung adenocarcinoma tissues and promotes migration and invasion in A549 cells. (A) Resistin protein expression in lung adenocarcinoma tissues (T) and paracarcinoma tissues (N) was detected by Western blot. (B) Correlation between MMP 2 protein expression and resistin protein expression in lung adenocarcinoma tissues. (C) A549 cells were seeded at 2000 cells/well in 96‐well plates and treated with different concentrations of resistin (0, 12.5, 25, 50, or 100 ng/mL) for 48 h. Cell proliferation was detected by the MTS assay. OD, optical density. (D) A549 cells were seeded on a 6‐well plate and incubated until confluence, then a cell‐free space was created by scraping. Migration was induced by treatment with different concentrations of resistin (0, 12.5, 25, 50, or 100 ng/mL) for 32 h. (E) 1 × 10 4 A549 cells were seeded in the upper chamber in 100 μl serum‐free medium with different concentrations of resistin (0, 12.5, 25, 50, 100 ng/ml), and 600 ?l 10% FBS RPMI 1640 medium was added to the lower chamber. For invasion assay, the upper insert was pre‐coated with growth factor‐reduced Matrigel. After 12h incubation, migrated cells were fixed for Transwell migration assay while invaded cells were fixed after 24 h incubation for Transwell invasion assay. (F) A549 cells were treated with or without 50 ng/mL resistin for 24 h. Total protein was isolated and expression of MMP 2 and Twist1 was detected by immunoblot assay. β‐Actin was used as an internal control. Data represent mean ± SD . n = 3; * P < 0.05

Article Snippet: Recombinant human resistin and resistin with His‐tag were purchased from ProSpec (Rehovot, Israel).

Techniques: Migration, Expressing, Western Blot, MTS Assay, Incubation, Invasion Assay, Transwell Migration Assay, Transwell Invasion Assay, Isolation, Control

Journal: Cancer Science

Article Title: Resistin facilitates metastasis of lung adenocarcinoma through the TLR 4/Src/ EGFR / PI 3K/ NF ‐κB pathway

doi: 10.1111/cas.13704

Figure Lengend Snippet: Toll‐like receptor 4 ( TLR 4) is the functional receptor of human resistin in A549 lung adenocarcinoma cells for migration and invasion. (A) A549 cells (1 × 10 4 ) were seeded in the upper Transwell chamber in serum‐free media and incubated with 50 ng/mL resistin after pretreatment with or without 5 μmol/L TAK ‐242 ( TLR 4 inhibitor). Migrated or invaded cells were photographed in at least five randomly chosen fields. A549 cells pretreated with or without 5 μmol/L TAK ‐242 for 1 h were treated with 50 ng/mL resistin for 24 h. The relative mRNA (B) and protein (C) expression of MMP 2 and Twist1 was quantitated. (D) A549 cells were seeded in a 6‐well plate and transfected with negative control (NC) mimic or TLR 4 si RNA at 70% confluence. After 24 h of transfection, cells were seeded in the upper chamber (1 × 10 4 cells per well) treated with or without 50 ng/mL resistin. Migrated or invaded cells were counted. (E) A549 cells were seeded in a 6‐well plate and transfected with NC mimic or TLR 4 si RNA at 70% confluence. After 24 h of transfection, cells were treated with or without 50 ng/mL resistin. After 24 h, total protein was isolated and expression of MMP 2, TLR 4, and Twist1 was detected. (F) A549 cells were treated with or without 50 ng/mL resistin for 24 h. Total protein was isolated and expression of TLR 4 was detected. (G) A549 cells were treated with or without 50 ng/mL resistin for 24 h. Expression of TLR 4 in A549 membrane was detected by flow cytometry. (H) A549 cells were treated with human recombinant resistin with His‐tag (500 ng/mL) for 1 h and then lysed and immunoprecipitated with agarose beads‐conjugated anti‐His mouse clonal antibody. IP, immunoprecipitation. (I) Five domains of TLR 4 and GST were purified and incubated with U937 cell lysates. Immunoprecipitation assay was carried out. Data represent mean ± SD . n = 3; * P < 0.05

Article Snippet: Recombinant human resistin and resistin with His‐tag were purchased from ProSpec (Rehovot, Israel).

Techniques: Functional Assay, Migration, Incubation, Expressing, Transfection, Negative Control, Isolation, Membrane, Flow Cytometry, Recombinant, Immunoprecipitation, Purification

Journal: Cancer Science

Article Title: Resistin facilitates metastasis of lung adenocarcinoma through the TLR 4/Src/ EGFR / PI 3K/ NF ‐κB pathway

doi: 10.1111/cas.13704

Figure Lengend Snippet: Epidermal growth factor receptor ( EGFR ) is involved in resistin‐induced migration and invasion of lung adenocarcinoma cells. (A) A549 cells (1 × 10 4 ) were seeded in the upper chamber in serum‐free media and incubated with 50 ng/mL resistin after pretreatment with or without 1 μmol/L erlotinib HC l ( EGFR inhibitor). Migrated or invaded cells were counted. (B,C) A549 cells pretreated with or without 1 μmol/L erlotinib HC l for 1 h were treated with 50 ng/mL resistin for 24 h. Relative mRNA (B) and protein (C) expression of MMP 2 and Twist1 was quantitated. (D) A549 cells were seeded in a 6‐well plate and transfected with negative control (NC) mimic or EGFR si RNA at 70% confluence. After 24 h of transfection, cells were seeded in the upper chamber (1 × 10 4 cells per well) treated with or without 50 ng/mL resistin. Migrated or invaded cells were counted. (E) A549 cells were seeded in a 6‐well plate and transfected with NC mimic or EGFR si RNA at 70% confluence. After 24 h of transfection, cells were treated with or without 50 ng/mL resistin. After 24 h, total protein was isolated and expression of MMP 2, EGFR , and Twist1 were detected. (F) A549 cells pretreated with or without 5 μmol/L TAK ‐242 and/or 10 μmol/L PP 1 (Src inhibitor) for 1 h were treated with 50 ng/mL resistin for 15 min. Total protein was isolated and expression of phosphorylated (p‐)Src, Src, p‐ EGFR , and EGFR was detected by Western blot

Article Snippet: Recombinant human resistin and resistin with His‐tag were purchased from ProSpec (Rehovot, Israel).

Techniques: Migration, Incubation, Expressing, Transfection, Negative Control, Isolation, Western Blot

Journal: Cancer Science

Article Title: Resistin facilitates metastasis of lung adenocarcinoma through the TLR 4/Src/ EGFR / PI 3K/ NF ‐κB pathway

doi: 10.1111/cas.13704

Figure Lengend Snippet: Phosphatidylinositol 3‐kinase ( PI 3K)/Akt/nuclear factor‐κB ( NF ‐κB) are the downstream signals mediating resistin‐induced migration and invasion of lung adenocarcinoma cells. (A) A549 cells (1 × 10 4 ) were seeded in the upper Transwell chamber in serum‐free media and incubated with 50 ng/mL resistin after pretreatment with or without 10 μmol/L LY 294002 ( PI 3K inhibitor) or 10 μmol/L BAY 11‐7082 ( NF ‐κB inhibitor). The migrated or invaded cells were counted. (B) A549 cells pretreated with or without 10 μmol/L LY 294002 or 10 μmol/L BAY 11‐7082 for 1 h were treated with 50 ng/mL resistin for 24 h. Total protein was isolated and expression of MMP 2 and Twist1 was detected by Western blot. (C) A549 cells pretreated with or without 5 μmol/L TAK ‐242, 10 μmol/L PP 1, 1 μmol/L erlotinib HC l, or 10 μmol/L LY 294002 for 1 h were treated with 50 ng/mL resistin for 15 min. Total protein was isolated and expression of phosphorylated (p‐)p85, p85, p‐Akt, and Akt was detected by Western blot. (D) A549 cells pretreated with or without 5 μmol/L TAK ‐242, 10 μmol/L PP 1, 1 μmol/L erlotinib HC l, 10 μmol/L LY 294002, or 10 μmol/L BAY 11‐7082 for 1 h were treated with 50 ng/mL resistin for 2 h. Cells were fixed and blocked and then incubated with primary antibody against p65 and Alexa Fluor 488‐conjugated secondary antibody. Stained cells were photographed with a fluorescence microscope. (E) A549 cells were seeded in a 6‐well plate and transfected with negative control mimic, Toll‐like receptor 4 ( TLR 4) si RNA , or epidermal growth factor receptor ( EGFR ) si RNA at 70% confluence. After 24 h of transfection, cells were treated with or without 50 ng/mL resistin. After 24 h, cells were fixed and blocked and then incubated with primary antibody against p65 and Alexa Fluor 488‐conjugated secondary antibody. Stained cells were photographed with a fluorescence microscope

Article Snippet: Recombinant human resistin and resistin with His‐tag were purchased from ProSpec (Rehovot, Israel).

Techniques: Migration, Incubation, Isolation, Expressing, Western Blot, Staining, Fluorescence, Microscopy, Transfection, Negative Control

Journal: Cancer Science

Article Title: Resistin facilitates metastasis of lung adenocarcinoma through the TLR 4/Src/ EGFR / PI 3K/ NF ‐κB pathway

doi: 10.1111/cas.13704

Figure Lengend Snippet: Schematic illustration of the molecular mechanism underlying the migration and invasion induced by resistin in A549 lung adenocarcinoma cells. EGFR , epidermal growth factor receptor; NF ‐κB, nuclear factor‐κB; TLR 4, Toll‐like receptor 4

Article Snippet: Recombinant human resistin and resistin with His‐tag were purchased from ProSpec (Rehovot, Israel).

Techniques: Migration

Journal: Journal of Translational Medicine

Article Title: Multi-omics analysis reveals a feedback loop amplifying immune responses in acute graft-versus-host disease due to imbalanced gut microbiota and bile acid metabolism

doi: 10.1186/s12967-024-05577-x

Figure Lengend Snippet: Granulocyte-like cells derived from patients with non-aGVHD exhibited elevated levels of RETN expression, suggesting a potential role for resistin in preventing aGVHD. A Heatmap display of the communication probability of each cluster within the combined dataset of PBMCs from patients undergoing allo-HSCT. B RETN and its corresponding receptor CAP1 were expresed in granulocyte-like cells across each sample. C Representative immunofluorescence staining of CD45 and resistin in the small intestine of control or aGVHD mouse model groups. Scale bar, 100 µm. D IL-1β concentrations were measured using ELISA assay in cell culture supernatants (n = 3) following various treatments involving LPS and resistin. E Venn diagrams illustrating the shared conserved downregulated genes among distinct lineages (group 1: neutrophils vs. macrophages vs. monocytes; group 2: cDC1 vs. cDC2 vs. migratory DC; group 3: plasmacytoid dendritic cell (pDC) vs. B cells; group 4: CD4 + T cells vs. CD8 + T cells vs. Treg; group 5: innate lymphoid cell (ILC) vs. γδ T cells vs. NK cells). F Summary of the resistin-driven immune regulation in the presence of LPS or other special conditions

Article Snippet: Following PMA stimulation, the culture medium was replaced with RPMI1640 supplemented with 10% FBS, 1 × P/S, and LPS (1, 10, or 100 ng/mL) (Cat# L8880, Salarbio) or recombinant human resistin (1, 10, and 100 ng/mL) (Cat# CJ48, novoprotein) for 24 h. The resulting cell-free supernatants were harvested and centrifuged at 500× g for 10 min.

Techniques: Derivative Assay, Expressing, Immunofluorescence, Staining, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Journal of Translational Medicine

Article Title: Multi-omics analysis reveals a feedback loop amplifying immune responses in acute graft-versus-host disease due to imbalanced gut microbiota and bile acid metabolism

doi: 10.1186/s12967-024-05577-x

Figure Lengend Snippet: A simplified schematic diagram illustrating the dysregulated gut microbiota and bile acid metabolism positive feedback loop, which exacerbates immune responses and exacerbates aGVHD. Improper treatment of patients undergoing allo-HSCT alters the gut microbiota and disrupts the imbalanced bile acid metabolism. Dysregulated bile acid metabolism results in reduced production of IL1RN, defensins, and resistin. Deficiency of defensins and resistin fails to inhibit harmful microorganisms, leading to increased production of proinflammatory cytokines and proteins, such as IL-17A, IL-1β, TNFα, IL-6, IL-12, and members of the S100A family. Additionally, it was determined that resistin can inhibit aGVHD by downregulating the expression of IL1B, DUSP1, and members of the AP-1 family

Article Snippet: Following PMA stimulation, the culture medium was replaced with RPMI1640 supplemented with 10% FBS, 1 × P/S, and LPS (1, 10, or 100 ng/mL) (Cat# L8880, Salarbio) or recombinant human resistin (1, 10, and 100 ng/mL) (Cat# CJ48, novoprotein) for 24 h. The resulting cell-free supernatants were harvested and centrifuged at 500× g for 10 min.

Techniques: Expressing

Journal: Acta Endocrinologica (Bucharest)

Article Title: RESISTIN EFFECT ON TELOMERASE GENE EXPRESSION IN GASTRIC CANCER CELL LINE AGS

doi: 10.4183/aeb.2016.145

Figure Lengend Snippet: Resistin effect on the cell viability. Cell viability performed by XTT assay, after triplicate cell plating, treatment, and incubation. The most effective dose of cell viability of AGS cell line was 10 ng/ml. Data are shown at the mean ± SEM.

Article Snippet: The medium was then replaced with serum-free medium containing different doses of human recombinant Resistin protein (Syd Labs, Japan) and cells were incubated for 12h, 24h and 48h for cell proliferation assay.

Techniques: XTT Assay, Incubation

Journal: Acta Endocrinologica (Bucharest)

Article Title: RESISTIN EFFECT ON TELOMERASE GENE EXPRESSION IN GASTRIC CANCER CELL LINE AGS

doi: 10.4183/aeb.2016.145

Figure Lengend Snippet: Resistin effect on hTERT mRNA in AGS gastric cancer cell line. AGS cells were treated with Resistin at 10 ng/mL and incubated for 6, 12 and 24h to examine telomerase gene expression by real time PCR. Data are represented as the mean ± SEM.

Article Snippet: The medium was then replaced with serum-free medium containing different doses of human recombinant Resistin protein (Syd Labs, Japan) and cells were incubated for 12h, 24h and 48h for cell proliferation assay.

Techniques: Incubation, Gene Expression, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Heparanase Interacts with Resistin and Augments Its Activity

doi: 10.1371/journal.pone.0085944

Figure Lengend Snippet: Lane 1, wash fraction. Lanes 2–4, elution fractions. Lane 5, molecular weight markers (in kDa). Resistin was identified in the 18 kDa band excised and subjected to MS analysis (arrow).

Article Snippet: A pure preparation of human recombinant resistin expressed in HEK 293 was a gift from Serono and was used in all assays.

Techniques: Molecular Weight

Journal: PLoS ONE

Article Title: Heparanase Interacts with Resistin and Augments Its Activity

doi: 10.1371/journal.pone.0085944

Figure Lengend Snippet: A : Lanes 1–6: co-immunprecipitation of resistin with either latent cMyc-heparanase or active heparanase imunoprecipitated with the indicated antibodies. Lane 7: a reference resisitin without co-immunprecipitation was run on the same gel. Immunoblot detection with rabbit anti resistin. The co-immunoprecipitated resistin is indicated by an arrow. B : Lane 1: a reference active heparanase without co-immunprecipitation. Lanes 2–4: co-immunprecipitation of resistin with active heparanase imunoprecipitated with the indicated antibodies. Immunoblot detection with rabbit anti heparanase. The co-immunoprecipitated active heparanase is indicated by an arrow. i.p- immunprecipitation; i.b- immunoblotting. C : Lane 1: a reference latent cmyc- heparanase without co-immunprecipitation. Lanes 2–5: co-immunprecipitation of resistin with cmyc-latent heparanase imunoprecipitated with the indicated antibodies (all run on the same gel). Immunoblot detection with mouse anti cMyc monoclonal antibody. The co-immunoprecipitated latent cMyc-heparanase is indicated by a red arrow.

Article Snippet: A pure preparation of human recombinant resistin expressed in HEK 293 was a gift from Serono and was used in all assays.

Techniques: Western Blot, Immunoprecipitation

Journal: PLoS ONE

Article Title: Heparanase Interacts with Resistin and Augments Its Activity

doi: 10.1371/journal.pone.0085944

Figure Lengend Snippet: A–C : ELISA of resistin with either latent cMyc-heparanase or active heparanase. A (left panel): coating layer: active heparanase; analyte: resistin; detection: anti resistin antibody. A (right panel): coating layer: resistin; analyte: active heparanase; detection: anti heparanase antibody. B (left panel): coating layer: cMyc latent heparanase; analyte: resistin; detection: anti resistin antibody. B (right panel): coating layer: resistin; analyte: cMyc latent heparanase; detection: anti cMyc antibody. C (left panel): coating layer: resistin in the absence (solid line) or presence of DTT (broken line); analyte: cMyc latent heparanase; detection: anti cMyc antibody. C (right panel): coating layer: cMyc latent heparanase; analyte: resistin untreated (solid line) or treated with DTT (broken line); detection: anti resistin antibody. Reagent background (O.D 630) obtained under the ELISA conditions and consisting of all layers of the ELISA except the analyte, was subtracted from each data point. Each data point represents the mean ±SE of triplicate wells. D (left panel): Competition of resistin binding (analyte) to cMyc latent heparanase (coat layer) by active heparanase. Resistin binding was measured without competitor (full circles, solid line), or in the presence of active heparanase (34 nM, squares, broken line), or leptin, a negative control (125 nM, diamonds, broken line). *p = 0.01 for resistin binding in the absence or presence of active heparanase. D (right panel): Competition on the binding of resistin (analyte, 200 ng/ml) to cMyc latent heparanase (coat layer) by active heparanase, presented as percent inhibition. Bound resisitin was detected by anti resistin antibodies. Reagent background (nonspecific signals, O.D 630) consisting of all layers of the ELISA except the analyte, was subtracted from each data point. Each data point represents the mean ±SE of triplicate wells.

Article Snippet: A pure preparation of human recombinant resistin expressed in HEK 293 was a gift from Serono and was used in all assays.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control, Inhibition

Journal: PLoS ONE

Article Title: Heparanase Interacts with Resistin and Augments Its Activity

doi: 10.1371/journal.pone.0085944

Figure Lengend Snippet: Shown are binding curves (top to bottom) of the following estimated resistin concentrations: 10, 6.25, 5, 3.3 and 2.5 nM.

Article Snippet: A pure preparation of human recombinant resistin expressed in HEK 293 was a gift from Serono and was used in all assays.

Techniques: Binding Assay

Journal: PLoS ONE

Article Title: Heparanase Interacts with Resistin and Augments Its Activity

doi: 10.1371/journal.pone.0085944

Figure Lengend Snippet: Cells were treated with PMA (100 nM, a ) or PMA with 10 ( b ), 20 ( c ), 40 ( d ), 80 ( e ), 160 ( f ) and 320 ( g ) ng/ml resistin. h . Oil red staining of the differentiated cells at the highest (320 ng/ml) concentration of resistin. Each experiment was repeated 3 times yielding similar results. A representative experiment is presented. i . Quantification of cell differentiation (oil red staining) in response to increased resistin concentrations. Each data point represents the mean ±SE of triplicate wells. *p = 0.001 for 20 vs. 160 ng/ml resistin.

Article Snippet: A pure preparation of human recombinant resistin expressed in HEK 293 was a gift from Serono and was used in all assays.

Techniques: Staining, Concentration Assay, Cell Differentiation

Journal: PLoS ONE

Article Title: Heparanase Interacts with Resistin and Augments Its Activity

doi: 10.1371/journal.pone.0085944

Figure Lengend Snippet: A, B : Enhancement of resistin (0.3 µg/ml) and PMA (100 nM) mediated differentiation of THP1 cells by latent cMyc-heparanase (1.25 µg/ml). Cells were treated with PMA in the absence ( a ), or presence of latent heparanase ( b ), resistin ( c ) or both ( d ). Cells were then subjected to Oil red staining to visualize oil droplets. Shown are representative photomicrographs at low (×4, A ) and high (×20, B ) magnifications. Each experiment was repeated 3 times yielding similar results. A representative experiment is presented. e . Quantification of cell differentiation (oil red staining) in response to resistin, heparanase, or both. Each data point represents the mean ±SE of triplicate wells. Counts (80) obtained for control cells treated with PMA alone were subtracted from all the treatment conditions shown in panel e . *p = 0.001 for resistin vs. resistin+heparanase. Figure 6 . C : Enhancement of resistin (50 ng/ml) and PMA (100 nM) mediated differentiation of THP1 cells by active heparanase. Dose response of heparanase is shown. Cells were treated with PMA in the absence ( a ) or presence of active heparanase (5 µg/ml; b ), resistin ( c ), or both resistin and heparanase (active heparanase 0.6 µg/ml, 1.25 µg/ml, 5 µg/ml, d–f respectively). Each experiment was repeated 3 times yielding similar results. A representative experiment is presented. e . Quantification of cell differentiation (oil red staining) shown in each panel. Each data point represents the mean ±SE of triplicate wells. **p = 0.001 for resistin vs. resistin+heparanase (5 µg/ml); *p = 0.01 for resistin vs. resistin+heparanase (1.2 µg/ml).

Article Snippet: A pure preparation of human recombinant resistin expressed in HEK 293 was a gift from Serono and was used in all assays.

Techniques: Staining, Cell Differentiation, Control