soluble cxcl16  (MedChemExpress)

Journal: International immunopharmacology

Article Title: MiR-625-5p is a potential therapeutic target in sepsis by regulating CXCL16/CXCR6 axis and endothelial barrier.

doi: 10.1016/j.intimp.2024.112508

Figure Lengend Snippet: Fig. 1. Predictive value of biomarkers and clinical variables on cumulative ICU survival in patients with sepsis or septic shock patients. Blood samples were collected from patients withsepsis or septic shock and healthy subjects (control) for qRT-PCR analysis of miR-625-5p. miR-625-5p was normalized with the level of h-SNORD44 (internal control) to determine the ratios, and the ratios of the control were arbitrarily set at 1. The relative levels of miR-625-5p (a) and CXCL16 (b) were detected by qRT-PCR. The levels of CXCL16 (c), SDC-1 (d), HS (e), and VE-cadherin (f) were detected by using ELISA. The red rulers in Figures a and d represent mean values with SDs, quantitative data was compared with one-way ANOVA between three groups. The blue rulers in Figures b, c, e, and f represent the medians with ranges, quantitative data was compared with Kruskal–Wallis analysis between three groups. ROC curve analysis of APACHE II score, SOFA score, miR-625-5p, CXCL16, SDC-1, PCT, and lactate at admission for the prediction of 28-day mortality (g). Kaplan–Meier survival estimates for all patients with sepsis or septic shock according to the respective level of miR-625-5p (miR-625-5p, cut-of: 25) (h). Correlations between biomarker levels and various clinical parameters (i–n). *P < 0.05, **P < 0.01.

Article Snippet: After reaching 80 %–90 % confluence, cells were treated with 5 and 10 μg/mL LPS (Sigma-Aldrich, L2880) for 6 or 12 h or soluble CXCL16 (sCXCL16; 0–20 ng/mL; MCE, HY-P72961) for 6 h.

Techniques: Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Biomarker Discovery

Journal: International immunopharmacology

Article Title: MiR-625-5p is a potential therapeutic target in sepsis by regulating CXCL16/CXCR6 axis and endothelial barrier.

doi: 10.1016/j.intimp.2024.112508

Figure Lengend Snippet: Fig. 3. miR-625-5p regulated CXCL16 transcription and expression. EA.hy926 cells were transfected with miR-625-5p mimic (100 nM) or negative control (miR- 625-5p mimic NC) for 48 h. qRT-PCR analysis of miRNA expression levels for miR-625-5p by transfection of miR-625-5p mimics (a). The relative level of CXCL16 was detected by qRT-PCR (b), CXCL16 supernatant levels were quantified by ELISA (c), whereas the expression levels in EA.hy926 cells were detected by Western blotting (d). Quantification of CXCL16 is illustrated in (e). *P < 0.05, **P < 0.01.

Article Snippet: After reaching 80 %–90 % confluence, cells were treated with 5 and 10 μg/mL LPS (Sigma-Aldrich, L2880) for 6 or 12 h or soluble CXCL16 (sCXCL16; 0–20 ng/mL; MCE, HY-P72961) for 6 h.

Techniques: Expressing, Transfection, Negative Control, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: International immunopharmacology

Article Title: MiR-625-5p is a potential therapeutic target in sepsis by regulating CXCL16/CXCR6 axis and endothelial barrier.

doi: 10.1016/j.intimp.2024.112508

Figure Lengend Snippet: Fig. 4. Inhibitor of miR-625-5p attenuated LPS-induced EA.hy926 cell barrier injury. EA.hy926 cells were transfected with the miR-625-5p inhibitor (50 nM) or negative control (micrOFF inhibitor NC) for 48 h and then exposed to LPS (5 and 10 µg/mL) for 6 h. The relative level of CXCL16 was detected by qRT-PCR (a), the expression levels of CXCL16 in the EA.hy926 cells were detected by Western blotting (b), and CXCL16 supernatant levels were quantified by ELISA (c). Quantification of CXCL16 is illustrated in (d). The effect of LPS and miR-625-5p mimic on the permeability of EA.hy926 cell were assessed using FITC-dextran and TEER methods (e, f). *P < 0.05, **P < 0.01.

Article Snippet: After reaching 80 %–90 % confluence, cells were treated with 5 and 10 μg/mL LPS (Sigma-Aldrich, L2880) for 6 or 12 h or soluble CXCL16 (sCXCL16; 0–20 ng/mL; MCE, HY-P72961) for 6 h.

Techniques: Transfection, Negative Control, Quantitative RT-PCR, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Permeability

Journal: International immunopharmacology

Article Title: MiR-625-5p is a potential therapeutic target in sepsis by regulating CXCL16/CXCR6 axis and endothelial barrier.

doi: 10.1016/j.intimp.2024.112508

Figure Lengend Snippet: Fig. 5. Effects of sCXCL16 on endothelial barrier integrity, glycocalyx, and tight junction in EA.hy926 cells. EA.hy926 cells were exposed to sCXCL16 (0, 2.5, 5, 10, and 20 ng/mL) treatment. Cell viability in EA.hy926 cells after treatment with sCXCL16 (a). Endothelial permeability was measured by using FITC- dextran and TEER 6 h after sCXCL16 treatment (b, c). The levels of HS, SDC-1, claudin5, occludin, and VE-cadherin were detected by Western blotting (d). EA.hy926 cells were divided into five groups: Group 1, control; Group 2, sCXCL16 2.5 ng/mL; Group 3, sCXCL16 5 ng/mL; Group 4, sCXCL16 10 ng/mL; Group 5, sCXCL16 20 ng/mL. Quantification of HS, SDC-1, claudin5, occludin, and VE-cadherin are shown in (e), *P < 0.05, vs. sCXCL16 0 (ng/mL) groups, **P < 0.01, vs sCXCL16 0 (ng/mL) groups. Immunofluorescence images of HS and SDC-1 in EA.hy926 cells (f, g) (magnifcation × 200, scale bar 150 µm), Fluorescence intensity analysis of h, i, respectively. * P < 0.05, **P < 0.01.

Article Snippet: After reaching 80 %–90 % confluence, cells were treated with 5 and 10 μg/mL LPS (Sigma-Aldrich, L2880) for 6 or 12 h or soluble CXCL16 (sCXCL16; 0–20 ng/mL; MCE, HY-P72961) for 6 h.

Techniques: Permeability, Western Blot, Control, Immunofluorescence, Fluorescence

Journal: International immunopharmacology

Article Title: MiR-625-5p is a potential therapeutic target in sepsis by regulating CXCL16/CXCR6 axis and endothelial barrier.

doi: 10.1016/j.intimp.2024.112508

Figure Lengend Snippet: Fig. 6. CXCL16 knockdown inhibited LPS-induced endothelial cell injury in vitro. EA.hy926 cells were transfected with CXCL16 siRNA (siCXCL16-1, 2, or 3) or negative control (siRNA NC) and CXCL16 expression was determined after 24 h. The relative level of CXCL16 were detected by qRT-PCR (a). EA.hy926 cells were divided into four groups: Group 1, control; Group 2, LPS; Group 3, LPS + siCXCL16; Group 4, siRNA NC. The cells in Group 3 were transfected with CXCL16 siRNA (siCXCL16), whereas cells in the other groups were transfected with negative control siRNA (siRNA NC). At 24 h post-transfection, the cells in Groups 2 and 3 were treated with 10 µg/mL LPS. Endothelial permeability was measured by using FITC- dextran and TEER 6 h after LPS treatment (b, c). The levels of HS, SDC-1, claudin5, occludin, and VE-cadherin were detected by Western blotting (d). Quantification of HS, SDC-1, claudin5, occludin, and VE-cadherin are shown in (e): *P < 0.05, vs. Control group; & P < 0.05, vs. LPS group. Immunofluorescence images of HS and SDC-1 in EA.hy926 cells (f, h; magnification, ×200; scale bar, 150 µm). Fluo rescence intensity analysis of g and i. *P < 0.05, **P < 0.01.

Article Snippet: After reaching 80 %–90 % confluence, cells were treated with 5 and 10 μg/mL LPS (Sigma-Aldrich, L2880) for 6 or 12 h or soluble CXCL16 (sCXCL16; 0–20 ng/mL; MCE, HY-P72961) for 6 h.

Techniques: Knockdown, In Vitro, Transfection, Negative Control, Expressing, Quantitative RT-PCR, Control, Permeability, Western Blot, Immunofluorescence

Journal: International immunopharmacology

Article Title: MiR-625-5p is a potential therapeutic target in sepsis by regulating CXCL16/CXCR6 axis and endothelial barrier.

doi: 10.1016/j.intimp.2024.112508

Figure Lengend Snippet: Fig. 7. Effects of sCXCL16 on CXCR6 in EA.hy926 cells. a–c: the EA.hy926 cells were exposed to sCXCL16 (0, 2.5, 5, 10, and 20 ng/mL) treatment. The relative levels of CXCR6 were detected by qRT-PCR (a), the expression levels of CXCR16 in EA.hy926 cells were detected by Western blotting (b), and the quantification of CXCR6 is illustrated in (c). *P < 0.05, **P < 0.01.

Article Snippet: After reaching 80 %–90 % confluence, cells were treated with 5 and 10 μg/mL LPS (Sigma-Aldrich, L2880) for 6 or 12 h or soluble CXCL16 (sCXCL16; 0–20 ng/mL; MCE, HY-P72961) for 6 h.

Techniques: Quantitative RT-PCR, Expressing, Western Blot

Journal: International immunopharmacology

Article Title: MiR-625-5p is a potential therapeutic target in sepsis by regulating CXCL16/CXCR6 axis and endothelial barrier.

doi: 10.1016/j.intimp.2024.112508

Figure Lengend Snippet: Fig. 8. CXCR6 knockout inhibited sCXCL16-induced EA.hy926 cell injury in vitro. EA.hy926 cells were transfected with CXCR6 siRNA (siCXCR6-1, 2, or 3) or negative control (siRNA NC) and CXCR6 expression was determined after 24 h. The relative level of CXCR6 were detected by qRT-PCR (a). EA.hy926 cells were divided into five groups: Group 1, control; Group 2, siCXCR6; Group 3, siCXCR6 + sCXCL16; Group 4, siCXCR6 NC; Group 5, sCXCL16 (20 ng/mL). The cells in Groups 2 and 3 were transfected with CXCR6 siRNA (siCXCR6), whereas the cells in the other groups were transfected with negative control siRNA (siRNA NC). At 24 h post-transfection, the cells in Groups 3 and 5 were treated with 20 ng/mL sCXCL16. Endothelial permeability was measured by using FITC- dextran and TEER 6 h after sCXCL16 treatment (b, c). The levels of HS, SDC-1, claudin5, occludin, and VE-cadherin were detected by Western blotting (d). Quantification of HS, SDC-1, claudin5, occludin, and VE-cadherin is illustrated in (e), * P < 0.05, vs Control group; # P < 0.05, vs. sCXCL16 (20 ng/mL) group. Immunofluorescence images of HS and SDC-1 in EA.hy926 cells (f, h; magnification, ×200; scale bar, 150 µm). Fluorescence intensity analysis of g and i. *P < 0.05, **P < 0.01.

Article Snippet: After reaching 80 %–90 % confluence, cells were treated with 5 and 10 μg/mL LPS (Sigma-Aldrich, L2880) for 6 or 12 h or soluble CXCL16 (sCXCL16; 0–20 ng/mL; MCE, HY-P72961) for 6 h.

Techniques: Knock-Out, In Vitro, Transfection, Negative Control, Expressing, Quantitative RT-PCR, Control, Permeability, Western Blot, Immunofluorescence, Fluorescence

Journal: International immunopharmacology

Article Title: MiR-625-5p is a potential therapeutic target in sepsis by regulating CXCL16/CXCR6 axis and endothelial barrier.

doi: 10.1016/j.intimp.2024.112508

Figure Lengend Snippet: Fig. 9. Mechanisms by which miR-625-5p disrupts lung endothelial barrier integrity. miR-625-5p level may be an effective biomarker for predicting 28-day mortality in patients with sepsis or septic shock. Furthermore, LPS-induced vascular endothelial hyper-permeability by regulating miR-625-5p/CXCL16/CXCR6 axis in sepsis. ① LPS increased miR‑625-5p expression in EA.hy926 cells; ② miR-625-5p positively regulated CXCL16 in EA.hy926 cells; ③ Treatment of EA.hy926 cells with LPS significantly increased CXCL16 release; ④ CXCL16 combines with CXCR6; ⑤ CXCR6 mediated the effects of CXCL16 on endothelial barrier dysfunction.

Article Snippet: After reaching 80 %–90 % confluence, cells were treated with 5 and 10 μg/mL LPS (Sigma-Aldrich, L2880) for 6 or 12 h or soluble CXCL16 (sCXCL16; 0–20 ng/mL; MCE, HY-P72961) for 6 h.

Techniques: Biomarker Discovery, Permeability, Expressing

Journal: Frontiers in Medicine

Article Title: CXCL16/CXCR6 axis arises as a potential peripheral biomarker of early COPD development – results from a pilot study

doi: 10.3389/fmed.2025.1636360

Figure Lengend Snippet: Enhanced circulating CXCL16 and CXCR6 expression on platelets and neutrophil-platelet aggregates in GOLD 1 patients. Plasma soluble CXCL16 levels (pg/mL) were measured by ELISA ( A ). Flow cytometry analysis of CXCR6-expressing platelets ( C ), and a representative dot plot ( D ). Flow cytometry analysis of CXCR6-expressing neutrophil-(CD16 + CD41 + ) ( F ) or eosinophil-platelet aggregates (CD16 – CD41 + ) ( H ). Results are presented as the percentage of positive (CXCR6 + ) platelets or leukocyte-platelet aggregates. Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the respective non-smoker group. † P < 0.05 or †† P < 0.01 relative to values in the normal LF smoker group. Correlations between the FEV1/FVC ratio and the plasma levels of CXCL16 ( B ), the percentage of CXCR6-expressing platelets ( E ), and the percentage of CXCR6-expressing neutrophil-platelet aggregates ( G ). FEV1, forced expiratory volume in the first second; FVC, forced vital capacity.

Article Snippet: Human plasma soluble CXCL16 was measured by an enzyme-linked immunosorbent assay (ELISA; DuoSet ® ELISA Kit, R&D Systems, Abingdon, United Kingdom–Catalog number: DY1164–Detection range: 15.6–1,000 pg/mL; sensitivity: 15.6 pg/mL; the specificity was evaluated by the manufacturer using several soluble factors tested at concentrations of 50 ng/mL, and none of these showed cross-reactivity or interference with the assay, supporting its high specificity for CXCL16).

Techniques: Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: Advanced Science

Article Title: LncRNA LINK‐ A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

doi: 10.1002/advs.202303341

Figure Lengend Snippet: LINK‐A overexpression reduces adaptive thermogenesis in HFD‐fed mice by remodeling the regional inflammatory microenvironment a) Heatmap of inflammatory factor mRNA expression levels in the mammary glands of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice detected by qRT‐PCR. per group n = 6. b–d) Representative images of H&E‐stained sections and IHC of mammary glands(b). The IL‐1β(c) or CXCL16(d) protein levels were analyzed using ImageJ. Scale bar: 50 µm. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, * p <0.05, ** p <0.01, **** p <0.0001. e–g) Representative images of H&E‐stained sections and IHC of scWAT(e). The IL‐1β(f) or CXCL16(g) protein levels were analyzed using ImageJ. Scale bar: 50 µm. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, **** p <0.0001. h–i) Mouse plasma concentrations of IL‐1β(h) and CXCL16(i) were measured by ELISA. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, **** p <0.0001. j) The protein levels of UCP1, P‐PKA, PKA, P‐HSL, and HSL in BMSCs‐derived beige adipocytes treatment using IL‐β or/and CXCL16 cytokines by immunoblot analysis, and the protein levels were quantified using ImageJ. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. k) Schematic diagram of the effect of inflammatory factors IL‐1β and CXCL16 on adipose thermogenesis. l–m) Following one week of dietary intervention, IL‐1β (l) and Cxcl16 (m) mRNA expression levels of the mammary gland in WT or LINK‐A KI mice were detected by qRT‐PCR. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, * p <0.05, ** p <0.01, **** p <0.0001.

Article Snippet: After successful induction of beige adipocytes, soluble IL‐1β inhibitor (VX‐765, MCE, 10 μ m ) or/and soluble CXCL16 inhibitor (GI254032, Selleck, 3 μ m ) was added to the medium, and cells were cultured for 24 h, followed by collecting cells for subsequent experiments.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Staining, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot

Journal: Advanced Science

Article Title: LncRNA LINK‐ A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

doi: 10.1002/advs.202303341

Figure Lengend Snippet: LINK‐A overexpression induces inflammatory factor expression by stabilizing HIF1α through the HFD‐induced HB‐EGF a) Schematic diagram of HRE site at CXCL16 promoters(up) and HIF1α binds the CXCL16 promotor region by HIF1α ChIP‐seq database analysis(down). b) CXCL16 ChIP‐qPCR primer sequences(blue) and HRE site sequences (yellow). c) The interaction between HIF1α and CXCL16 promoter in MCF‐10A was verified by ChIP‐qPCR assay. Data presented as mean ± SEM, pooled data from three independent experiments, unpaired t‐test, **** p <0.0001. d) IL‐1β ChIP‐qPCR primer sequences(blue) and HRE site sequences(yellow). e) The interaction between HIF1α and IL‐1β promoter in MCF‐10A was verified by ChIP‐qPCR assay. Data presented as mean ± SEM, pooled data from three independent experiments, unpaired t‐test, ** p <0.01. f) Immunoblot analysis of HIF1α protein levels in the mammary gland of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice. The HIF1α protein was quantified using ImageJ. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, **** p <0.0001. g) qRT‐PCR analysis of Hif1α mRNA in mammary glands of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, **** p <0.0001. h) The interaction between HIF1α and CXCL16 promoter (Pro) in MCF‐10A was verified by a Dual‐Luciferase reporter assay. The CXCL16 Pro mutant (mut) site contains HRE(b). Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ** p <0.01. i) The interaction between HIF1α and IL‐1β promoter in MCF‐10A was verified by a Dual‐Luciferase reporter assay. The IL‐1β Pro mut site contains HRE(d). Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, *** p <0.001. j) The mRNA levels of CXCL16 , IL‐1β in MCF‐10A empty vector (EV) or HIF1α overexpression (OV‐HIF1α) cells were detected by qRT‐PCR. Data presented as mean ± SEM, pooled data from three independent experiments, two‐way ANOVA, **** p <0.0001. k) The mRNA levels of CXCL16 , IL‐1β in MCF‐10A shControl (shCtrl) or shHIF1α (shHIF1α#1, shHIF1α#2) cells by qRT‐PCR. Data presented as mean ± SEM, pooled data from three independent experiments, two‐way ANOVA, ns = no significance, **** p <0.0001. l) qRT‐PCR analysis of Hb‐egf mRNA in mammary glands of ND‐WT, ND‐KI, HFD‐WT, and HFD‐KI mice. Data presented as mean ± SEM, per group n = 6, one‐way ANOVA, ns = no significance, * p <0.05, ** p <0.01, **** p <0.0001. m) The protein level of HIF1α in MCF‐10A EV or LINK‐A overexpression (OV‐ LINK‐A ) cells with or without HB‐EGF stimulation was detected by immunoblot analysis, and the HIF1α protein was quantified using ImageJ. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, ** p <0.01, *** p <0.001, **** p <0.0001. n) The mRNA level of HIF1α in MCF‐10A EV or LINK‐A overexpression cells with or without HB‐EGF stimulation was detected by qRT‐PCR. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance. o,p) The concentrations of IL‐1β(o), and CXCL16(p) in the culture medium were measured by ELISA. Data presented as mean ± SEM, pooled data from six independent experiments, one‐way ANOVA, ns = no significance, * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. q) Schematic diagram of LINK‐A promotes the expression of IL‐1β and CXCL16 by stabilizing their transcription factors HIF1α.

Article Snippet: After successful induction of beige adipocytes, soluble IL‐1β inhibitor (VX‐765, MCE, 10 μ m ) or/and soluble CXCL16 inhibitor (GI254032, Selleck, 3 μ m ) was added to the medium, and cells were cultured for 24 h, followed by collecting cells for subsequent experiments.

Techniques: Over Expression, Expressing, ChIP-sequencing, ChIP-qPCR, Western Blot, Quantitative RT-PCR, Luciferase, Reporter Assay, Mutagenesis, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Advanced Science

Article Title: LncRNA LINK‐ A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

doi: 10.1002/advs.202303341

Figure Lengend Snippet: Activation of the LINK‐A /HB‐EGF/HIF1α pathway in mammary cells reduces adipocyte thermogenesis a) Schematic representation of bone marrow mesenchymal stem cells (BMSCs)‐derived beige adipocytes treated with MCF‐10A cells conditioned media: LINK‐A overexpressing MCF‐10A and control cells were stimulated with HB‐EGF, and the conditioned media was obtained to treat beige adipocytes. b) The protein levels of UCP1, P‐PKA, PKA, P‐HSL, and HSL in adipocytes treated with different conditioned media by immunoblot analysis, and the protein levels were quantified using ImageJ. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, ** p <0.01, *** p <0.001, **** p <0.0001. c–f) The mRNA levels of thermogenic genes in adipocytes treated with different conditioned media by qRT‐PCR. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, ** p <0.01, *** p <0.001, **** p <0.0001. g) Schematic representation of BMSCs‐derived beige adipocytes treated with MCF‐10A cells conditioned media: IL‐1β inhibitor or/and CXCL16 inhibitors add to the conditioned media of HB‐EGF‐stimulated LINK‐A overexpression MCF‐10A cells, respectively, and the conditioned media was obtained to treat beige adipocytes. h) The protein levels of UCP1, P‐PKA, PKA, P‐HSL, and HSL in adipocytes treated with different conditioned media by immunoblot analysis, and the protein levels were quantified using ImageJ. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, ** p <0.01, *** p <0.001, **** p <0.0001. i–l) The mRNA levels of thermogenic genes in adipocytes treated with different conditioned media by qRT‐PCR. Data presented as mean ± SEM, pooled data from three independent experiments, one‐way ANOVA, ns = no significance, * p <0.05, *** p <0.001, **** p <0.0001. m) Schematic diagram of LINK‐A affecting adipocyte thermogenesis.

Article Snippet: After successful induction of beige adipocytes, soluble IL‐1β inhibitor (VX‐765, MCE, 10 μ m ) or/and soluble CXCL16 inhibitor (GI254032, Selleck, 3 μ m ) was added to the medium, and cells were cultured for 24 h, followed by collecting cells for subsequent experiments.

Techniques: Activation Assay, Derivative Assay, Control, Western Blot, Quantitative RT-PCR, Over Expression

Journal: Advanced Science

Article Title: LncRNA LINK‐ A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

doi: 10.1002/advs.202303341

Figure Lengend Snippet: ASO drug inhibiting LINK‐A attenuates obesity and metabolic disorders in mice a) Schematic diagram of the mouse mammary gland in situ LINK‐A LNAs injection strategy. b) LINK‐A levels in mice mammary glands injected with LINK‐A LNAs in situ for two weeks by qRT‐PCR. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01. c) Body weight changes in HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment. Data presented as mean ± SEM, per group n = 6, two‐way ANOVA, ns = no significance, **** p <0.0001. d,e) GTT(d) and ITT(e) of HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment. Data presented as mean ± SEM, per group n = 6, two‐way ANOVA, * p <0.05, *** p <0.001. f) The HIF1α protein levels in the mammary gland of HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment were detected by immunoblot analysis, and the HIF1α protein levels were quantified using ImageJ. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01. g,h) The plasma concentrations of IL‐1β(g), and CXCL16(h) in HFD‐KI mice with LINK‐A LNAs or Scr LNAs treatment were measured by ELISA. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01. i–k) Representative images of H&E‐stained sections and IHC of the mammary gland (nipple)(i), mammary gland (duct)(j), and scWAT(k), and the IL‐1β or CXCL16 protein levels were analyzed using ImageJ. Scale bar: 50 µm. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01. l) HFD‐KI treated with LINK‐A LNAs or Scr LNAs for two weeks were placed at 4 °C, 48 h, and their protein levels of UCP1, P‐PKA, PKA, P‐HSL, HSL in scWAT were detected by immunoblot analysis, and the protein levels were quantified using ImageJ. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01. m) HFD‐KI treated with LINK‐A LNAs or Scr LNAs for two weeks were placed at 4 °C, 48 h, and their mRNA levels of thermogenic genes in scWAT were detected by qRT‐PCR. Data presented as mean ± SEM, per group n = 6, Mann–Whitney U‐test, ** p <0.01.

Article Snippet: After successful induction of beige adipocytes, soluble IL‐1β inhibitor (VX‐765, MCE, 10 μ m ) or/and soluble CXCL16 inhibitor (GI254032, Selleck, 3 μ m ) was added to the medium, and cells were cultured for 24 h, followed by collecting cells for subsequent experiments.

Techniques: In Situ, Injection, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining

Journal: Advanced Science

Article Title: LncRNA LINK‐ A Remodels Tissue Inflammatory Microenvironments to Promote Obesity

doi: 10.1002/advs.202303341

Figure Lengend Snippet: The LINK‐A expression was positively correlated with the expression of inflammatory cytokines in overweight people a–e) Representative images of H&E‐stained sections and IHC of breast tissue microarrays(a) of patients with overweight, and the inflammatory factor levels were analyzed using ImageJ. Scale bar: 50 µm. Data presented as mean ± SEM, n = 12, chi‐square test, ns = no significance, **** p <0.0001. f–i) Correlation between IL‐1β , CXCL16 , LINK‐A and BMI. Data presented as mean ± SEM, n = 45, Pearson chi‐square test. * p <0.05, ** p <0.01, *** p <0.001. j–m) Correlation between BMI, IL‐1β , CXCL16 , LINK‐A , and HIF1α protein. Data presented as mean ± SEM, n = 24, Pearson chi‐square test. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001. n) Article General Mechanism Diagram.

Article Snippet: After successful induction of beige adipocytes, soluble IL‐1β inhibitor (VX‐765, MCE, 10 μ m ) or/and soluble CXCL16 inhibitor (GI254032, Selleck, 3 μ m ) was added to the medium, and cells were cultured for 24 h, followed by collecting cells for subsequent experiments.

Techniques: Expressing, Staining

Journal: Cancer Science

Article Title: Wnt5a‐Ror2 signaling in mesenchymal stem cells promotes proliferation of gastric cancer cells by activating CXCL16–CXCR6 axis

doi: 10.1111/cas.12871

Figure Lengend Snippet: Conditioned media from mesenchymal stem cells (MSCs) promote proliferation of MKN45‐Luc cells in a manner dependent on Ror2 expression in MSCs. (a) Conditioned media from MSCs promotes proliferation of MKN45‐Luc cells. MKN45‐Luc cells were cultured in the presence or absence of MSC‐conditioned media. Luciferase activities were measured at the indicated time points. Data are expressed as mean ± SD ( n = 3). * P < 0.05; ** P < 0.001, t ‐test. (b) MKN45‐Luc cells were cultured in the presence of conditioned media from MSCs pretreated with either ctrl siRNA or three different siRNAs against Ror2 (#1, #2, #3). After 9 days in culture, luciferase activities were measured. Data are expressed as mean ± SD ( n = 3). * P < 0.05; ** P < 0.001, t ‐test. (c) MSCs were transfected with either ctrl or Ror2 (#2) siRNA. After 6 days in culture, conditioned media were collected and subjected to chemokine array analysis. Boxed spots (B4) indicate protein levels of CXCL16, which decreased significantly and reproducibly following suppressed expression of Ror2 . Other spots with different intensities between si‐Ctrl and si‐ Ror2 groups include C4 (interleukin [IL]‐8), C9 (CCL2), and E6 (CXCL12). A1, A10, and G1 represent reference spots. (d) Expression of CXCL16 is downregulated by suppressed expression of Ror2 in MSCs. Mesenchymal stem cells were transfected with either ctrl or Ror2 siRNAs. After 6 days in culture, mRNA levels of CXCL16 (left panel) and Ror2 (right panel) were measured by quantitative RT‐PCR analyses. (e) MSCs were transfected with either ctrl or Ror2 (#1) siRNA. After 6 days in culture, conditioned media were collected to measure relative amounts of CXCL16 protein by ELISA. Data are expressed as mean ± SD ( n = 3). * P < 0.05, t ‐test.

Article Snippet: In some experiments, MKN45‐Luc cells were treated with soluble recombinant human CXCL16 (PeproTech, Oak Park, CA, USA) at a final concentration of 1.0 ng/mL.

Techniques: Expressing, Cell Culture, Luciferase, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Cancer Science

Article Title: Wnt5a‐Ror2 signaling in mesenchymal stem cells promotes proliferation of gastric cancer cells by activating CXCL16–CXCR6 axis

doi: 10.1111/cas.12871

Figure Lengend Snippet: Expression of CXCL16 in mesenchymal stem cells (MSCs) is required for the ability of MSCs to promote proliferation of MKN45‐Luc cells in coculture. (a) Recombinant CXCL16 (rCXCL16) promotes proliferation of MKN45‐Luc cells. MKN45‐Luc cells were cultured in the absence (vehicle) or presence of 1 ng/mL rCXCL16, and luciferase activities were measured at the indicated time points. Data are expressed as mean ± SD ( n = 3). ** P < 0.001, t ‐test. (b,c) Suppressed expression of CXCL16 in MSCs. MSCs were transfected with either ctrl or CXCL16 siRNA. After 6 days in culture, mRNA levels of CXCL16 were measured by quantitative RT‐PCR analyses (b). Conditioned media from the siRNA‐treated MSCs were collected to measure relative amounts of CXCL16 protein by ELISA (c). (d,e) MKN45‐Luc cells were cultured singly or cocultured with siRNA‐treated MSCs either directly (d) or indirectly (e). Luciferase activities were measured at the indicated time points. Data are expressed as mean ± SD ( n = 3). ** P < 0.001, t ‐test. (f,g) Effect of neutralizing antibody against CXCL16 on proliferation of MKN45‐Luc cells cocultured with MSCs. MKN45‐Luc cells were cocultured with MSCs either directly (f) or indirectly (g) in the presence of anti‐CXCL16 neutralizing antibody or control IgG. Luciferase activities were measured at the indicated time points. Data are expressed as mean ± SD ( n = 3). ** P < 0.001, t ‐test.

Article Snippet: In some experiments, MKN45‐Luc cells were treated with soluble recombinant human CXCL16 (PeproTech, Oak Park, CA, USA) at a final concentration of 1.0 ng/mL.

Techniques: Expressing, Recombinant, Cell Culture, Luciferase, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control