Journal: Cell Death & Disease

Article Title: Elevated fatty acid β-oxidation by leptin contributes to the proinflammatory characteristics of fibroblast-like synoviocytes from RA patients via LKB1-AMPK pathway

doi: 10.1038/s41419-023-05641-2

Figure Lengend Snippet: A RA-FLS were stimulated with vehicle, serum from RA patient and HC with or without anti-leptin antibody (500 ng/mL), together with serum from RA patient and isotype antibody (500 ng/mL). Cells were harvested at 24 h and mRNA expression of the indicated genes was determined with real-time PCR analysis ( n = 5). B Measurement of CPT-1A, CPT-1B and CPT-1C mRNA expressions in RA-FLS by real-time PCR (left, n = 5). RA-FLS were treated with vehicle, serum from HC with or without leptin, serum from rheumatoid arthritis with or without leptin antibody, isotype antibody, and levels of CPT-1A were determined by western blot ( n = 4). C Expression of leptin receptor was assessed in RA-FLS treated by vehicle, serum from HC and RA patient with real-time PCR analysis ( n = 5), western blot and immunofluorescence assay (scale bar = 200 μm, n = 4). D CPT-1A was tested with real-time PCR in RA-FLS stimulated by leptin (100 ng/mL), anti-leptin antibody (500 ng/mL) and isotype antibody (500 ng/mL) for 24 h, respectively ( n = 5). CPT-1A protein expression was also detected at 48 h after indicated stimulations ( n = 4). Data are means ± SEM of three independent experiments, statistical significance was determined as ** P < 0.01, * P < 0.05, ns means no significance compared with control.

Article Snippet: In order to block the effect of leptin, RA-FLS were handled by serum from HC or RA patients with or without human leptin antibody (anti-leptin, R&D Systems) and mouse IgG1 isotype control (R&D Systems).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Control

Journal: Cell Death & Disease

Article Title: Elevated fatty acid β-oxidation by leptin contributes to the proinflammatory characteristics of fibroblast-like synoviocytes from RA patients via LKB1-AMPK pathway

doi: 10.1038/s41419-023-05641-2

Figure Lengend Snippet: A Protein–protein interactions (PPI) among leptin, leptin receptor and major metabolic pathways was consturcted. Only the PPI with the confidence of >0.4 was kept. B AMPK total and phosphorylated protein at T172 (pAMPK), LKB1 and CAMKK protein levels were compared in RA-FLS treated in the presence or absence of leptin by western blot ( n = 4). C Assessment of LKB1 protein level in RA-FLS processed with vehicle, anti-leptin antibody and isotype antibody by western blot ( n = 4). D AMPK total and pAMPK in NC or down-regulation of LKB1 with siRNA in the presence or absence of leptin was detected at 48 h ( n = 4). E CPT-1A gene ( n = 5) and protein ( n = 4) levels in RA-FLS stimulated by leptin with or without compound C (CC, 5 μM) was evaluated. Data are means ± SEM of three independent experiments, statistical significance was determined as ** P < 0.01, * P < 0.05, *** P < 0.001, **** P < 0.0001, ns means no significance compared with control.

Article Snippet: In order to block the effect of leptin, RA-FLS were handled by serum from HC or RA patients with or without human leptin antibody (anti-leptin, R&D Systems) and mouse IgG1 isotype control (R&D Systems).

Techniques: Protein-Protein interactions, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Digital microfluidic assay for protein detection

doi: 10.1073/pnas.1323998111

Figure Lengend Snippet: IL-6 and leptin microfluidic immunoassays; 50% human sera was spiked with recombinant IL-6 (5 nM), leptin (600 nM), or IL-6 and leptin mixed together. Each sample was analyzed using both anti–IL-6 (A) and anti-leptin (B) chips to demonstrate specificity of our microfluidic sandwich immunoassay approach. Titrating concentrations of IL-6 were also analyzed using anti–IL-6 chips under the same conditions to evaluate dynamic range and sensitivity (C). Detection of the bound cytokine of interest was performed using anti–IL-6– or anti-leptin–conjugated beads. Bound beads were optically quantified, and normalized bead counts were calculated by dividing by the number of beads observed to nonspecifically be bound in a control channel (no antigen, 50% human serum) performed in parallel. A dynamic range of at least six orders or magnitude and a limit of detection of 50 pM was observed.

Article Snippet: Recombinant human IL-6 (206-IL), anti–IL-6 antibodies (AF-206-NA, MAB206), and anti-leptin antibodies (MAB398, BAM398) were purchased from R&D Systems.

Techniques: Recombinant, Control

Journal: Annals of medicine

Article Title: Human adipose-derived stem cells promote migration of papillary thyroid cancer cell via leptin pathway.

doi: 10.1080/07853890.2024.2419990

Figure Lengend Snippet: Figure 1. Leptin receptor (Ob-R) antagonist reduced the number of metastatic lung nodules and the diameter of nodules. (A) Procedure of animal experiment. Nude mice received venous transplantation of TPC-1 cells. To block OB-R, 1 mg/kg of Allo-aca was administrated to the animal per day. (B) Gross morphology of mice lungs showed nodules. Allo-aca reduced the number of pulmonary nodules. (C) Lung nodules were diagnosed as metastatic tumour under the light microscope. Allo-aca reduced the diameter of the nodules. (D) The lung nodules were examined by Western blot of thyroglobulin (TG). The nodules in the lungs were TG -positive, while the lung tissue was TG -negative. This result confirmed that the lung nodules were from thyroid carci noma. (E–H) Allo-aca increased the level of serum T3, and had no significant effect on the level of T4, TSH and the body weight of the animal. Experiments were repeated at least five times. Data are expressed as mean ± standard deviation. * p < 0.05, *** p < 0.001, ns: not statistically significant.

Article Snippet: the cell or tissue samples were pretreated with RiPa lysis buffer and protease inhibitors (both from Beyotime institute of Biotechnology). total proteins were determined using a multimode microplate reader (type Biotek synergy h1, agilent, inc.) and separated by 10% sDs-PaGe gel electrophoresis and transferred to PVDF membranes. the PVDF membranes were blocked with Bsa (sigma-aldrich) and then hybridized with antibodies, including the following: mouse monoclonal igG of leptin receptor (Ob-R) antibody (R&D systems, catalog# MaB867, diluted 1:500), rabbit polyclonal MMP-2 (cell signaling technology, catalog# 4022, diluted 1:1000), MMP-9 antibodies (signaling technology, catalog# 3852, diluted 1:1000), rabbit monoclonal thyroglobulin antibody (abcam, catalog# ab243094, diluted 1:1000), rabbit polyclonal GaPDh antibody (abcam, catalog# ab9485, diluted 1:1000), goat anti-mouse igG (abcam, catalog# ab6789, diluted 1:2000), goat anti-rabbit igG (abcam, catalog# ab205718, diluted 1:2000). the PVDF membranes were incubated with a developing solution and developed in the Molecular imager Gel Doc XR system (Bio-Rad laboratories, inc.).

Techniques: Transplantation Assay, Blocking Assay, Light Microscopy, Western Blot, Standard Deviation

Journal: Annals of medicine

Article Title: Human adipose-derived stem cells promote migration of papillary thyroid cancer cell via leptin pathway.

doi: 10.1080/07853890.2024.2419990

Figure Lengend Snippet: Figure 4. The conditioned medium of ADSCs upregulated the level of leptin receptor (Ob-R) in TPC-1 and BCPAP cells. (A) The concentration of leptin in the medium of ADSCs was ~3-fold higher than that in the medium of TPC-1 and BCPAP cells, approx imately 50 pg/ml. (B) In vitro experiments showed that ADSC-CM increased the level of leptin receptor (Ob-R) in the TPC-1/BCPAP cells. Experiments were repeated at least five times. Data are expressed as mean ± standard deviation. *p < 0.05, ***p < 0.001.

Article Snippet: the cell or tissue samples were pretreated with RiPa lysis buffer and protease inhibitors (both from Beyotime institute of Biotechnology). total proteins were determined using a multimode microplate reader (type Biotek synergy h1, agilent, inc.) and separated by 10% sDs-PaGe gel electrophoresis and transferred to PVDF membranes. the PVDF membranes were blocked with Bsa (sigma-aldrich) and then hybridized with antibodies, including the following: mouse monoclonal igG of leptin receptor (Ob-R) antibody (R&D systems, catalog# MaB867, diluted 1:500), rabbit polyclonal MMP-2 (cell signaling technology, catalog# 4022, diluted 1:1000), MMP-9 antibodies (signaling technology, catalog# 3852, diluted 1:1000), rabbit monoclonal thyroglobulin antibody (abcam, catalog# ab243094, diluted 1:1000), rabbit polyclonal GaPDh antibody (abcam, catalog# ab9485, diluted 1:1000), goat anti-mouse igG (abcam, catalog# ab6789, diluted 1:2000), goat anti-rabbit igG (abcam, catalog# ab205718, diluted 1:2000). the PVDF membranes were incubated with a developing solution and developed in the Molecular imager Gel Doc XR system (Bio-Rad laboratories, inc.).

Techniques: Concentration Assay, In Vitro, Standard Deviation

Journal: Annals of medicine

Article Title: Human adipose-derived stem cells promote migration of papillary thyroid cancer cell via leptin pathway.

doi: 10.1080/07853890.2024.2419990

Figure Lengend Snippet: Figure 5. The neutralizing antibody to leptin reduced the invasion and migration of the TPC-1 and BCPAP cells. ADSC-CM upreg ulated MMP-2 in TPC-1 and BCPAP cells, and the effect was attenuated by the neutralizing antibody to leptin. (A) Addition of 10 μg/mL NAB of leptin to ADSC-CM conditioned medium attenuated TPC-1 and BCPAP invasion. (B) Leptin NAB also attenuated conditioned medium-promoted TPC-1/BCPAP migration. Experiments were repeated at least five times. Data are expressed as mean ± standard deviation. *p < 0.05, ***p < 0.001.

Article Snippet: the cell or tissue samples were pretreated with RiPa lysis buffer and protease inhibitors (both from Beyotime institute of Biotechnology). total proteins were determined using a multimode microplate reader (type Biotek synergy h1, agilent, inc.) and separated by 10% sDs-PaGe gel electrophoresis and transferred to PVDF membranes. the PVDF membranes were blocked with Bsa (sigma-aldrich) and then hybridized with antibodies, including the following: mouse monoclonal igG of leptin receptor (Ob-R) antibody (R&D systems, catalog# MaB867, diluted 1:500), rabbit polyclonal MMP-2 (cell signaling technology, catalog# 4022, diluted 1:1000), MMP-9 antibodies (signaling technology, catalog# 3852, diluted 1:1000), rabbit monoclonal thyroglobulin antibody (abcam, catalog# ab243094, diluted 1:1000), rabbit polyclonal GaPDh antibody (abcam, catalog# ab9485, diluted 1:1000), goat anti-mouse igG (abcam, catalog# ab6789, diluted 1:2000), goat anti-rabbit igG (abcam, catalog# ab205718, diluted 1:2000). the PVDF membranes were incubated with a developing solution and developed in the Molecular imager Gel Doc XR system (Bio-Rad laboratories, inc.).

Techniques: Migration, Standard Deviation

Journal: Annals of medicine

Article Title: Human adipose-derived stem cells promote migration of papillary thyroid cancer cell via leptin pathway.

doi: 10.1080/07853890.2024.2419990

Figure Lengend Snippet: Figure 6. Neutralizing antibodies against leptin slightly downregulated cell proliferation without significant effect on cell viability. (A) Addition of leptin NAB to ADSC-CM slightly reduced the stimulatory effect of conditioned medium on TPC-1 proliferation. (B) Addition of NAB to the conditioned medium had no significant effect on cell viability of TPC-1 cells. (C) NAB of leptin had no significant effect on BCPAP proliferation. (D) NAB of leptin had no significant effect on BCPAP viability. Experiments were repeated at least five times. Data are expressed as mean ± standard deviation. *p < 0.05, ns: not statistically significant.

Article Snippet: the cell or tissue samples were pretreated with RiPa lysis buffer and protease inhibitors (both from Beyotime institute of Biotechnology). total proteins were determined using a multimode microplate reader (type Biotek synergy h1, agilent, inc.) and separated by 10% sDs-PaGe gel electrophoresis and transferred to PVDF membranes. the PVDF membranes were blocked with Bsa (sigma-aldrich) and then hybridized with antibodies, including the following: mouse monoclonal igG of leptin receptor (Ob-R) antibody (R&D systems, catalog# MaB867, diluted 1:500), rabbit polyclonal MMP-2 (cell signaling technology, catalog# 4022, diluted 1:1000), MMP-9 antibodies (signaling technology, catalog# 3852, diluted 1:1000), rabbit monoclonal thyroglobulin antibody (abcam, catalog# ab243094, diluted 1:1000), rabbit polyclonal GaPDh antibody (abcam, catalog# ab9485, diluted 1:1000), goat anti-mouse igG (abcam, catalog# ab6789, diluted 1:2000), goat anti-rabbit igG (abcam, catalog# ab205718, diluted 1:2000). the PVDF membranes were incubated with a developing solution and developed in the Molecular imager Gel Doc XR system (Bio-Rad laboratories, inc.).

Techniques: Standard Deviation

Journal: Annals of medicine

Article Title: Human adipose-derived stem cells promote migration of papillary thyroid cancer cell via leptin pathway.

doi: 10.1080/07853890.2024.2419990

Figure Lengend Snippet: Figure 8. Hypothesis diagram. Adipose tissue acts on thyroid cancer cells by secreting leptin. Leptin upregulates the level of the leptin receptor (Ob-R) on the surface of thyroid cancer cells and up-modulates the level of MMP-2 by activating the leptin signal ling pathway, which may be an important mechanism by which obesity promotes the metastasis of thyroid cancer cells.

Article Snippet: the cell or tissue samples were pretreated with RiPa lysis buffer and protease inhibitors (both from Beyotime institute of Biotechnology). total proteins were determined using a multimode microplate reader (type Biotek synergy h1, agilent, inc.) and separated by 10% sDs-PaGe gel electrophoresis and transferred to PVDF membranes. the PVDF membranes were blocked with Bsa (sigma-aldrich) and then hybridized with antibodies, including the following: mouse monoclonal igG of leptin receptor (Ob-R) antibody (R&D systems, catalog# MaB867, diluted 1:500), rabbit polyclonal MMP-2 (cell signaling technology, catalog# 4022, diluted 1:1000), MMP-9 antibodies (signaling technology, catalog# 3852, diluted 1:1000), rabbit monoclonal thyroglobulin antibody (abcam, catalog# ab243094, diluted 1:1000), rabbit polyclonal GaPDh antibody (abcam, catalog# ab9485, diluted 1:1000), goat anti-mouse igG (abcam, catalog# ab6789, diluted 1:2000), goat anti-rabbit igG (abcam, catalog# ab205718, diluted 1:2000). the PVDF membranes were incubated with a developing solution and developed in the Molecular imager Gel Doc XR system (Bio-Rad laboratories, inc.).

Techniques:

Journal: Annals of medicine

Article Title: Human adipose-derived stem cells promote migration of papillary thyroid cancer cell via leptin pathway.

doi: 10.1080/07853890.2024.2419990

Figure Lengend Snippet: Figure 7. (A,B) The ADSCs conditioned medium upregulated the MMP-2 levels of TPC-1/BCPAP. NAB of leptin partially counter acted the increase in MMP-2 levels. Experiments were repeated at least five times. Data are expressed as mean ± standard devia tion. *p < 0.05, **p < 0.01.

Article Snippet: the cell or tissue samples were pretreated with RiPa lysis buffer and protease inhibitors (both from Beyotime institute of Biotechnology). total proteins were determined using a multimode microplate reader (type Biotek synergy h1, agilent, inc.) and separated by 10% sDs-PaGe gel electrophoresis and transferred to PVDF membranes. the PVDF membranes were blocked with Bsa (sigma-aldrich) and then hybridized with antibodies, including the following: mouse monoclonal igG of leptin receptor (Ob-R) antibody (R&D systems, catalog# MaB867, diluted 1:500), rabbit polyclonal MMP-2 (cell signaling technology, catalog# 4022, diluted 1:1000), MMP-9 antibodies (signaling technology, catalog# 3852, diluted 1:1000), rabbit monoclonal thyroglobulin antibody (abcam, catalog# ab243094, diluted 1:1000), rabbit polyclonal GaPDh antibody (abcam, catalog# ab9485, diluted 1:1000), goat anti-mouse igG (abcam, catalog# ab6789, diluted 1:2000), goat anti-rabbit igG (abcam, catalog# ab205718, diluted 1:2000). the PVDF membranes were incubated with a developing solution and developed in the Molecular imager Gel Doc XR system (Bio-Rad laboratories, inc.).

Techniques: