Journal: Research Square

Article Title: Efferocytosis is restricted by axon guidance molecule EphA4 via ERK/Stat6/Mertk signaling following brain injury

doi: 10.21203/rs.3.rs-3079466/v1

Figure Lengend Snippet: (A) Uniform Manifold Approximation and Projection (UMAP) plot showing different cell clusters and their cell-specific annotation in the injured cortex at 1dpi (B) Dot plot of efferocytosis genes expressed by each cluster type. (C-F) Feature maps highlighting the expression across clusters of Mertk (C), Gas 6 (D), Pros1 (E), and Stat6 (F).

Article Snippet: On day 5, bone marrow-derived macrophages (BMDMS) were supplemented with fresh RPMI media or pretreated with 1 μg/ml of Escherichia coli O111:B4 LPS (Sigma Aldrich, St. Louis, MO) for 4 hours, 0.5 μg/ml of mouse recombinant HMGB1 (ThermoFisher Scientific) for 4 hours, 5 μg/ml of EphA4-Fc or Fc-control clustered using 1.7 μg/ml α-Fc (Sino Biological, Wayne, PA) for 1 hour, 25 μM of ERK inhibitor (FR18020R, Cayman chemicals) for 4 hours, 5 μM of Mertk inhibitor (UNC2025, Cayman Chemicals) for 4 hours, or 250 nM of Stat6 inhibitor (AS1517499, Cayman Chemicals) for 4 hours.

Techniques: Expressing

Journal: Research Square

Article Title: Efferocytosis is restricted by axon guidance molecule EphA4 via ERK/Stat6/Mertk signaling following brain injury

doi: 10.21203/rs.3.rs-3079466/v1

Figure Lengend Snippet: Peripheral EphA4 deficiency promotes P-ERK/P-Stat6/Mertk signaling. A-C) mRNA expression of the efferocytosis receptor (Mertk, A) and its ligands (Gas6, B) and (Pros1, C) in the contralateral and ipsilateral cortex of WT +WT BMCs and WT +KO BMCs mice at 3dpi. N=5–6 mice/group. Ns = non-significant; *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001. Two-way ANOVA followed by Šídák’s multiple comparisons test. D) Western blot analysis shows increased Mertk, ERK, and Stat6 phosphorylation in the ipsilateral cortex of chimeric WT +KO BMCs mice. E-H) Representative images showing peripheral-derived GFP+ cells expressing Mertk (purple) and P-ERK (E, F) and P-Stat6 (G, H) in WT +WT BMCs and WT +KO BMCs mice.

Article Snippet: On day 5, bone marrow-derived macrophages (BMDMS) were supplemented with fresh RPMI media or pretreated with 1 μg/ml of Escherichia coli O111:B4 LPS (Sigma Aldrich, St. Louis, MO) for 4 hours, 0.5 μg/ml of mouse recombinant HMGB1 (ThermoFisher Scientific) for 4 hours, 5 μg/ml of EphA4-Fc or Fc-control clustered using 1.7 μg/ml α-Fc (Sino Biological, Wayne, PA) for 1 hour, 25 μM of ERK inhibitor (FR18020R, Cayman chemicals) for 4 hours, 5 μM of Mertk inhibitor (UNC2025, Cayman Chemicals) for 4 hours, or 250 nM of Stat6 inhibitor (AS1517499, Cayman Chemicals) for 4 hours.

Techniques: Expressing, Western Blot, Phospho-proteomics, Derivative Assay

Journal: Research Square

Article Title: Efferocytosis is restricted by axon guidance molecule EphA4 via ERK/Stat6/Mertk signaling following brain injury

doi: 10.21203/rs.3.rs-3079466/v1

Figure Lengend Snippet: EphA4-null BMDMs show enhanced efferocytosis that is regulated by ERK/ Stat6/Mertk. A-I) EphA4 deletion enhances the efferocytosis efficiency of BMDMS in vitro . A-H) Representative images showing the engulfment of the pHrodo-stained apoptotic (but not live) Jurkat cells (red) by GFP+ untreated WT (A-D) and EphA4 KO (E-H) BMDMS. I) Quantification of the efferocytosis efficiency of WT and EphA4 KO BMDMS after stimulation with LPS and HMGB1 for 4 hours. J) Efferocytosis of EphA4 KO BMDMSs is mediated by the blockade of forward EphA4, not reverse ephrin signals. Treatment of WT and EphA4 KO BMDMS with clustered EphA4-FC to activate reverse ephrin signals did not reduce the efferocytosis of EphA4 KO BMDMS. K-M) mRNA expression of Mertk (K), Gas6 (L), and Pros1 (M) with and without engulfment of apoptotic Jurkat cells. EphA4 KO BMDMSs have higher expression of Mertk and Gas6 than WT. N) The use of Mertk inhibitor reduced efferocytosis of both WT and EphA4 KO BMDMS; however, Stat6 and ERK inhibitors selectively reduced efferocytosis in EphA4 KO BMDMS. O) Stat6 inhibitor reduced Mertk and Gas6 expression, and ERK inhibitor reduced Gas6 expression in KO BMDMS engulfing apoptotic Jurkat cells. P) Suggested pathway for the regulation of efferocytosis by EphA4. N=5–6 mice/group. Ns = non-significant; *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001. Two-way ANOVA followed by Šídák’s multiple comparisons test (I-N) or one-way ANOVA followed by Tukey’s multiple comparisons test (O).

Article Snippet: On day 5, bone marrow-derived macrophages (BMDMS) were supplemented with fresh RPMI media or pretreated with 1 μg/ml of Escherichia coli O111:B4 LPS (Sigma Aldrich, St. Louis, MO) for 4 hours, 0.5 μg/ml of mouse recombinant HMGB1 (ThermoFisher Scientific) for 4 hours, 5 μg/ml of EphA4-Fc or Fc-control clustered using 1.7 μg/ml α-Fc (Sino Biological, Wayne, PA) for 1 hour, 25 μM of ERK inhibitor (FR18020R, Cayman chemicals) for 4 hours, 5 μM of Mertk inhibitor (UNC2025, Cayman Chemicals) for 4 hours, or 250 nM of Stat6 inhibitor (AS1517499, Cayman Chemicals) for 4 hours.

Techniques: In Vitro, Staining, Expressing

Journal: Cancers

Article Title: EWSR1-WT1 Target Genes and Therapeutic Options Identified in a Novel DSRCT In Vitro Model

doi: 10.3390/cancers13236072

Figure Lengend Snippet: MERTK inhibitor UNC2025 affects DSRCT tumor expansion in vitro. ( A ) Barplot depicting qPCR results of relative MERTK expression in OV-054, JN-DSRCT-1, and huSI organoids, normalized to GAPDH . ( B ) Graph depicting cell viability of OV-054 DSRCT, JN-DSRCT-1, and huSI organoids, upon administration of 0, 25, 50, 100, 200, and 400 nM UNC2025. ( C ) Graph depicting IC50 of UNC2025 on both OV-054 DSRCT and JN-DSRCT-1 cells ( D ) Representative pictures of OV-054 DSRCT, JN-DSRCT-1, and huSI organoids in vitro, days 0 and 7, after administration of 0, 25, 50, 100, 200, and 400 nM UNC2025 (10× objective).

Article Snippet: OV-054 DSRCT cells, JN-DSRCT-1 cells, and control human small intestinal (huSI) organoids were incubated with different concentrations of MERTK inhibitor UNC2025 (Selleckchem, #S7576, Houston, TX, USA): 25, 50, 100, 200, or 400 nM was added to the culture medium.

Techniques: In Vitro, Expressing

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