Journal: Science Advances

Article Title: NLRP3 inflammasome constrains liver regeneration through impairing MerTK-mediated macrophage efferocytosis

doi: 10.1126/sciadv.adq5786

Figure Lengend Snippet: ( A ) ELISA analysis of serum IL-6 level ( n = 3 per group). ( B ) ELISA analysis of serum TNF-α level ( n = 3 per group). ( C and D ) Representative flow cytometry analysis plots and the quantification of Ly6C hi or Ly6C lo hepatic macrophages 36 hours after PHx ( n = 3 per group). ( E ) mRNA expression of Ly6C hi or Ly6C lo macrophage markers in isolated hepatic macrophages ( n = 3 per group). iNOS, inducible nitric oxide synthase; Arg-1, arginase-1. ( F ) RNA sequencing (RNA-seq) analysis of isolated hepatic macrophages 36 hours after PHx. Heatmap showed the proinflammatory (M1), reparative (M2), and efferocytosis gene expression profiles in Nlrp3 Δ mye mice and Nlrp3 fl/fl mice. ( G ) Bubble chart showing the top 20 of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of the significant up-regulated genes in Nlrp3 Δ mye mice. PPAR, peroxisome proliferator–activated receptor. ( H and I ) mRNA expression of efferocytosis genes in isolated hepatic macrophages 36 hours after PHx ( n = 3 per group). ( J ) Representative fluorescent images and quantification of engulfing ACs (TUNEL) by hepatic macrophages (CD68) in situ from Nlrp3 Δ mye mice and Nlrp3 fl/fl mice 36 hours after PHx (scale bar, 50 μm) ( n = 5 per group). ( K ) Flow cytometry analysis of MerTK expression in isolated hepatic macrophages 36 hours after PHx ( n = 3 per group). ( L ) Representative fluorescent images and quantification of engulfing ACs (pHrodo labeled, green) by BMDMs (F-actin labeled, red) isolated from Nlrp3 Δ mye mice and Nlrp3 fl/fl mice (scale bar, 50 μm) ( n = 5 per group). ( M ) mRNA expression of MerTK and HGF in isolated hepatic macrophages after cocultured with ACs ( n = 3 per group). Data are shown as means ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001. h, hours.

Article Snippet: For MerTK inhibition, UNC2025 (TargetMol, no. T7007) was administrated intraperitoneally to a concentration of 50 mg/kg 2 hours before and 24 hours after PHx.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Isolation, RNA Sequencing Assay, TUNEL Assay, In Situ, Labeling

Journal: Science Advances

Article Title: NLRP3 inflammasome constrains liver regeneration through impairing MerTK-mediated macrophage efferocytosis

doi: 10.1126/sciadv.adq5786

Figure Lengend Snippet: The NLRP3 inflammasome exhibited robust activation in macrophages during the initial phase of liver regeneration following 70% PHx. Blocking macrophage NLRP3 significantly enhanced liver regeneration, whereas overexpression of NLRP3 impaired it after PHx. Deficiency in Nlrp3 promoted MerTK-mediated efferocytosis, thereby inducing a pro-reparative Ly6C lo phenotype in macrophages. This image was drawn by the authors.

Article Snippet: For MerTK inhibition, UNC2025 (TargetMol, no. T7007) was administrated intraperitoneally to a concentration of 50 mg/kg 2 hours before and 24 hours after PHx.

Techniques: Activation Assay, Blocking Assay, Over Expression

Journal: Frontiers in Immunology

Article Title: Expression of TAM-R in Human Immune Cells and Unique Regulatory Function of MerTK in IL-10 Production by Tolerogenic DC

doi: 10.3389/fimmu.2020.564133

Figure Lengend Snippet: PROS1 enhances LPS-inducible IL-10 secretion in MerTK high moDC. (A) IL-10 was quantified by ELISA from supernatants of moDC or moDC-Dex stimulated by LPS (100 ng/ml) and/or PROS1 (10 μg/ml) ( n = 10). Statistical significance was determined with a Wilcoxon rank-sum test. (B) IL-10 was quantified by ELISA from supernatants of moDC-Dex incubated during 48 h with LPS, PROS1, and/or UNC2025 (1 μM) ( n = 5). Statistical significance was determined with paired t -test (** p < 0.01; * p < 0.05; ns, not significant).

Article Snippet: Cells were seeded at 1 · 10 6 cells/ml and treated with 100 ng/ml LPS (Invivogen), 10 μg/ml PROS1, and/or 1 μM UNC2025 (Selleckchem) or dimethylsulfoxide (DMSO) (vehicle).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation

Journal: Molecular cancer therapeutics

Article Title: MERTK mediates intrinsic and adaptive resistance to AXL-targeting agents

doi: 10.1158/1535-7163.MCT-17-1239

Figure Lengend Snippet: (A, B, C, D, F)HNSCC and TNBC cell lines were treated with vehicle (DMSO), R428, UNC2025, or R428+UNC2025 and relative cell numbers were determined after 72 hours. UM-SCC1, MDAMB231, and NSCLC cell lines were transfected with AXL siRNA (siAXL, siAXL10, siAXL12) or non-targeting siRNA (siNT) and then treated with vehicle or UNC2025 for 72 hours prior to determination of relative cell numbers. (E) NSCLC cell lines were cultured at low density and treated with vehicle (DMSO), R428, UNC2025, or R428+UNC2025 for 10 days. Colonies were stained and counted. *p<0.05, **p<0.01, ***p<0.001, NS=not significant.

Article Snippet: UNC2025 (MERTK inhibitor) was synthesized as previously described ( 26 ) or purchased from Selleck Chemicals or Apexbio.

Techniques: Transfection, Cell Culture, Staining

Journal: Molecular cancer therapeutics

Article Title: MERTK mediates intrinsic and adaptive resistance to AXL-targeting agents

doi: 10.1158/1535-7163.MCT-17-1239

Figure Lengend Snippet: (A, B)UM-SCC1 and MDAMB231 cells were treated with vehicle (DMSO), R428, UNC2025, or R428+UNC2025 for 24 hours. (C) H1299 cells were transfected with AXL siRNA (siAXL10 or siAXL12) or non-targeting siRNA (siNT) and cells were treated with vehicle or UNC2025 for one hour. Expression of the indicated proteins was assessed in cell lysates by immunoblot. α-Tubulin is shown as a loading control. The data shown are representative of 3 independent experiments.

Article Snippet: UNC2025 (MERTK inhibitor) was synthesized as previously described ( 26 ) or purchased from Selleck Chemicals or Apexbio.

Techniques: Transfection, Expressing, Western Blot, Control

Journal: Molecular cancer therapeutics

Article Title: MERTK mediates intrinsic and adaptive resistance to AXL-targeting agents

doi: 10.1158/1535-7163.MCT-17-1239

Figure Lengend Snippet: Subcutaneous xenografts were generated in nude mice using (A, D) the TNBC MDAMB231 cell line, (B) the HNSCC UW-SCC1 patient-derived xenograft (PDX), or (C) the HNSCC UM-SCC1 cell line. Tumor-bearing mice were treated with vehicle, 25mg/kg R428, 25 mg/kg UNC2025, or R428+UNC2025 administered twice daily via oral gavage. MERTK expression was assessed in tumor cell lysates by immunoblot. α-Tubulin was used as a loading control. (A) MERTK expression was also detected in MDAMB231 tumors using immunohistochemistry. (B, C, D) Tumor volume was measured (n=8 per group). (C, D) pS6rp and Ki67 were detected in tumor sections using IHC. Mean values and standard errors are shown. *p<0.05, **p<0.01, NS=not significant.

Article Snippet: UNC2025 (MERTK inhibitor) was synthesized as previously described ( 26 ) or purchased from Selleck Chemicals or Apexbio.

Techniques: Generated, Derivative Assay, Expressing, Western Blot, Control, Immunohistochemistry