Journal: EBioMedicine

Article Title: Pro-inflammatory monocyte profile in patients with major depressive disorder and suicide behaviour and how ketamine induces anti-inflammatory M2 macrophages by NMDAR and mTOR

doi: 10.1016/j.ebiom.2019.10.063

Figure Lengend Snippet: Ketamine induces a M2c-like phenotype in monocyte-derived macrophages with increased levels of MERTK, CD163, and intermediate levels of CD64 while reducing the response to LPS. Monocyte-derived macrophages were differentiated for 7 days in the presence or absence of ketamine (0.1, 1 and 10 µM), and the percentage of (a) MERTK, (b) CD163, (c) CD206 and (d) CD64 positive CD11b + macrophages was analysed by flow cytometry. Macrophage polarization controls were performed using dexamethasone (0.1 µM) for M2c, IL-4 (40 ng/mL) for M2a, and LPS (1 ng/mL) plus IFN-γ (50 ng/mL) for M1. Representative and independent data are shown. (e-i) To analyse the response to an inflammatory stimulus, ketamine-induced macrophages were stimulated for 24h with 1 ng/mL of LPS. The activation markers (e) CD80 and (f) HLADR were evaluated by flow cytometry and (g) TNF-α, (h) IL-6 and (i) IL-10 production was assessed by ELISA. Each dot represents an independent donor and pooled data were graphed. One-way ANOVA test was performed and statistical significance is denoted as * p < 0.05; ** p < 0.01; *** p < 0.001. Untreated condition: Untd; dexamethasone: DEX.

Article Snippet: The phenotype and activation of macrophages were characterized by cell surface staining employing the appropriate combination of directly conjugated antibodies against human CD11b-APC/Cy7 (BioLegend Cat # 101225, RRID: AB_830641), CD64-PE/Cy7 (BioLegend Cat # 305021, RRID: AB_2561583), CD163-PerCP/Cy5.5 (BioLegend Cat # 333625, RRID: AB_2,650629), CD206-AlexaFluor 488 (BioLegend Cat # 321113, RRID: AB_571874), CD14-PE (BioLegend Cat # 325605, RRID: AB_830678), HLA-DR-FITC (BioLegend Cat # 980402, RRID: AB_2616625), CD80-PE (BioLegend Cat # 305207, RRID: AB_314,503), and MERTK-APC (R&D Systems Cat # FAB8912A RRID:AB_357213) along with its control isotype IgG1-APC (R&D Systems, Cat # IC002A).

Techniques: Derivative Assay, Flow Cytometry, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: EBioMedicine

Article Title: Pro-inflammatory monocyte profile in patients with major depressive disorder and suicide behaviour and how ketamine induces anti-inflammatory M2 macrophages by NMDAR and mTOR

doi: 10.1016/j.ebiom.2019.10.063

Figure Lengend Snippet: NMDAR antagonist MK-801, but not the AMPAR antagonist NBQX, induces a similar M2 profile as ketamine, and this phenotype is completely abolished by the inhibition of the mTOR pathway. Monocyte-derived macrophages were differentiated for 7 days in the presence or absence of the NMDAR antagonist MK-801 (1 and 10 µM) or AMPAR antagonist NBQX (1 and 10 µM), and the percentage of (a) MERTK and (b) CD206 was analysed for M2 polarization by flow cytometry. Representative histograms and independent data are shown. Rapamycin (0.01–1 nM), added from day 0, was used to evaluate the role of the mTOR pathway in macrophage polarization after 7 days of culture. Viable CD11b + cells were analysed for the expression of (c) MERTK, (d) CD206, (e) CD64, and (f) CD163. Each experimental condition includes at least 4 independent donors. Pooled data were graphed and one-way ANOVA test was performed accordingly. Statistical significance is denoted as * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The phenotype and activation of macrophages were characterized by cell surface staining employing the appropriate combination of directly conjugated antibodies against human CD11b-APC/Cy7 (BioLegend Cat # 101225, RRID: AB_830641), CD64-PE/Cy7 (BioLegend Cat # 305021, RRID: AB_2561583), CD163-PerCP/Cy5.5 (BioLegend Cat # 333625, RRID: AB_2,650629), CD206-AlexaFluor 488 (BioLegend Cat # 321113, RRID: AB_571874), CD14-PE (BioLegend Cat # 325605, RRID: AB_830678), HLA-DR-FITC (BioLegend Cat # 980402, RRID: AB_2616625), CD80-PE (BioLegend Cat # 305207, RRID: AB_314,503), and MERTK-APC (R&D Systems Cat # FAB8912A RRID:AB_357213) along with its control isotype IgG1-APC (R&D Systems, Cat # IC002A).

Techniques: Inhibition, Derivative Assay, Flow Cytometry, Expressing

Journal: Clinical and Translational Medicine

Article Title: Chromium (VI)‐induced ALDH1A1/EGF axis promotes lung cancer progression

doi: 10.1002/ctm2.1136

Figure Lengend Snippet: Cr(VI)‐induced overexpression of ALDH1A1 maintains self‐renewal of CrT/TICs. (A) Cell sorting for ALDH1A1 High and ALDH1A1 Low CrT cells. CrT cells were stained with ALDEFLUOR kit and PI. ALDH1A1 High cells: AF top 10%, PI (–); ALDH1A1 Low cells: AF bottom 10%, PI (–). AF: ALDEFLUOR Fluorescence. (B) ALDH1A1 High and ALDH1A1 Low CrT cells were lysed for immunoblot analyses with the indicated antibodies. (C) Reactive oxygen species (ROS) levels were detected by DCFH‐DA staining in ALDH1A1 Low and ALDH1A1 High CrT cells exposed with or without Cr (VI). Data represent the mean ± SD of triplicate experiments. ** p < .001. (D) In vitro limiting dilution assays on ALDH1A1 High and ALDH1A1 Low CrT cells. ** p < .001. (E) Tumoursphere formation assays using ALDH1A1 High and ALDH1A1 Low CrT cells. (F) CrT/TICs with or without Dox‐inducible ALDH1A1 shRNA were treated with or without Dox and lysed for immunoblot analyses with the indicated antibodies. (G) In vitro limiting dilution assays on CrT/TICs cells with or without doxycycline (Dox)‐inducible ALDH1A1 shRNA. ** p < .001. (H) Tumoursphere formation assays using CrT/TICs with or without Dox‐inducible ALDH1A1 shRNA. (I) CrT/TICs with Dox‐inducible ALDH1A1 shRNA were subcutaneously implanted in the left side of mice. (J) CrT/TICs with Dox‐inducible ALDH1A1 shRNA were orthotopically implanted in the lung of mice. (Top) Representative BLIs of lung orthotopic tumours with or without Dox treatment for 50 days. (Bottom) Quantification of BLIs every 10 days. Data are presented as the mean ± SD from five mice. ** P < .001. (K) Kaplan–Meier survival curves for indicated mice. (L) Immunohistochemical (IHC) staining was performed with antibody against ALDH1A1. Scale bar, 20 μm. (M) IHC staining was performed with antibodies against Ki‐67, CD133, and CD44. Scale bar, 20 μm. (N) ALDH1A1 activity were detected in CrT/TICs with the indicated concentration of A37. Data represent the mean ± SD of triplicate experiments. * p < .01, *** p < .0001. (O) In vitro limiting dilution assays on CrT cells treated with or without A37 (50 μM). *** p < .0001. (P) Tumoursphere formation assays using CrT cells treated with or without A37 (50 μM). (Q) CrT/TICs were subcutaneously implanted in the left side of mice. (R) CrT/TICs were orthotopically implanted in the lung of mice. (Top) Representative BLIs of lung orthotopic tumours with or without A37 treatment for 50 days. (Bottom) Quantification of BLIs every 10 days. Data are presented as the mean ± SD from five mice. ** P < .001. (S) Kaplan–Meier survival curves for indicated mice. (T) IHC staining was performed with antibodies against Ki‐67, CD133, and CD44. Scale bar, 20 μm

Article Snippet: DACH1 siRNA (sc‐77089), ABCB5 siRNA (sc‐89856), MERTK siRNA (sc‐37127), KLF4 siRNA (sc‐35480), SOX2 siRNA (sc‐38408), EGF siRNA (sc‐39416), and ALDH1A1 siRNA (sc‐41442) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Over Expression, FACS, Staining, Fluorescence, Western Blot, In Vitro, shRNA, Immunohistochemical staining, Immunohistochemistry, Activity Assay, Concentration Assay

Journal: Clinical and Translational Medicine

Article Title: Chromium (VI)‐induced ALDH1A1/EGF axis promotes lung cancer progression

doi: 10.1002/ctm2.1136

Figure Lengend Snippet: Cr(VI) induces ALDH1A1 expression through KLF4. (A) CrT cells transfected with siRNAs (50 nM) targeting KLF4, DACH1, ABCB5, MERTK, SOX2 or EGF for 72 h and were lysed for immunoblot analyses with the indicated antibodies. (B) CrT cells transfected with or without EGF siRNA (50 nM, 72 h) were lysed for ELISA analyses for detecting secreted EGF levels in the culturing media. (C) CrT cells transfected with siRNAs (50 nM) targeting KLF4, DACH1, ABCB5, MERTK, SOX2 or EGF for 72 h and were lysed for qRT‐PCR analysis of ALDH1A1 mRNA expression levels. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (D) ALDH1A1 High and ALDH1A1 Low CrT cells were lysed for immunoblot analyses with the indicated antibodies. (E) ALDH1A1 Low CrT cells transfected with or without Flag‐KLF4 for 72 h were lysed for immunoblot analysis with the indicated antibodies. ALDH1A1 Low CrT cells transfected with or without Flag‐ALDH1A1 were lysed for immunoblot analyses with the indicated antibodies. (F) ALDH1A1 Low CrT cells transfected with or without KLF4 siRNA (50 nM) for 72 h were lysed for immunoblot analysis with the indicated antibodies. ALDH1A1 High CrT cells transfected with or without ALDH1A1 siRNA were lysed for immunoblot analyses with the indicated antibodies. (G) Schematic image represents the KLF4 binding sequence within the ALDH1A1 transcriptional regulation region. (H) Luciferase reporter assays were performed in BEAS‐2B and CrT cells transfected with pGL‐3.0 vector containing ALDH1A1 WT or mutant promoter. Data represent the mean ± SD of triplicate experiments. ** p < .001. (I) CrT cells with or without KLF4 depletion and BEAS‐2B cells with or without expression of Flag‐KLF4 were transfected with a luciferase reporter gene under the control of the ALDH1A1 promoter for 24 h. Luciferase reporter assays were performed. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (J) BEAS‐2B cells, CrT cells, and CrT/TICs were used for ChIP‐qPCR analysis of the ALDH1A1 promoter with the indicated antibody. Data are presented as the mean ± SD of triplicate experiments. * P < .01, ** P < .001. (K) CrT cells with or without KLF4 depletion were used for the detection of ALDH1A1 activity by flow cytometry. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (L) Tumoursphere formation assays using ALDH1A1 High CrT cells transfected with or without KLF4 siRNA. (M) In vitro limiting dilution assays on ALDH1A1 High CrT cells transfected with or without KLF4 siRNA. ** p < .001. (N) In vitro limiting dilution assays on ALDH1A1 Low CrT cells transfected with or without Flag‐KLF4. ** p < .001. (O) Tumoursphere formation assays using ALDH1A1 Low CrT cells transfected with or without Flag‐KLF4

Article Snippet: DACH1 siRNA (sc‐77089), ABCB5 siRNA (sc‐89856), MERTK siRNA (sc‐37127), KLF4 siRNA (sc‐35480), SOX2 siRNA (sc‐38408), EGF siRNA (sc‐39416), and ALDH1A1 siRNA (sc‐41442) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Expressing, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Binding Assay, Sequencing, Luciferase, Plasmid Preparation, Mutagenesis, Control, ChIP-qPCR, Activity Assay, Flow Cytometry, In Vitro

Journal: Clinical and Translational Medicine

Article Title: Chromium (VI)‐induced ALDH1A1/EGF axis promotes lung cancer progression

doi: 10.1002/ctm2.1136

Figure Lengend Snippet: CrT/TIC‐secreted EGF activates EGFR signalling and promotes LUSC cell growth. (A) HCC95 and H226 cells incubated with a conditioned medium or co‐cultured with the indicated cells were lysed for immunoblot analysis with the indicated antibodies; HCC95 and H226 cells co‐cultured with CrT/TICs transfected with or without KLF4 siRNA were lysed for immunoblot analyses with the indicated antibodies. (B) HCC95 and H226 cells incubated with CrT/TIC‐derived conditioned medium for 12 h in the presence or the absence of human recombinant truncated EGF or EGF L26G were lysed for immunoblot analysis with the indicated antibodies; HCC95 and H226 cells co‐cultured with CrT/TICs transfected with or without KLF4 siRNA were lysed for immunoblot analyses with the indicated antibodies. (C) HCC95 and H226 cells incubated with CrT/TIC‐derived conditioned medium for 12 h in the presence or the absence of EGF‐neutralising antibodies were lysed for immunoblot analyses with the indicated antibodies. (D) HCC95 and H226 cells co‐cultured with CrT/TICs with or without ALDH1A1 depletion were lysed for immunoblot analyses with the indicated antibodies. (E) HCC95 and H226 cells co‐cultured with CrT/TICs transfected with or without KLF4 siRNA were lysed for immunoblot analyses with the indicated antibodies. (F) HCC95 and H226 cells co‐cultured with CrT/TICs pretreated with or without A37 were lysed for immunoblot analyses with the indicated antibodies. (G) HCC95 and H226 cells incubated with conditional medium derived from ALDH1A1 Low CrT or ALDH1A1 High CrT were lysed for immunoblot analyses with the indicated antibodies. (H) Growth curves of HCC95 and H226 cells cultured with BEAS‐2B‐, CrT‐, and CrT/TIC‐derived conditioned medium. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (I) Growth curves of HCC95 and H226 cells cultured with CrT/TIC‐derived conditioned medium pretreated with truncated EGF or EGF L26G. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (J) Growth curves for the HCC95 and H226 cells cultured with CrT/TICs‐derived conditional medium pretreated with or without anti‐EGF antibody. Data represent the mean ± SD of triplicate experiments. ** p < .001. (K) Growth curves of HCC95 and H226 cells cultured with the indicated conditioned medium derived from CrT/TICs with or without ALDH1A1 depletion. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (L) Growth curves of HCC95 and H226 cells cultured with the indicated conditioned medium derived from CrT/TICs with or without A37 treatment. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (M) HCC95 and H226 cells co‐cultured with CrT/TICs pretreated with or without U0126 were lysed for immunoblot analyses with the indicated antibodies. (N) HCC95 and H226 cells co‐cultured with CrT/TICs pretreated with or without PD98 were lysed for immunoblot analyses with the indicated antibodies. (O) Growth curves of HCC95 and H226 cells with or without U0126 treatment cultured with the indicated conditioned medium derived from CrT/TICs. Data are presented as the mean ± SD of triplicate experiments. ** P < .001. (P) Growth curves of HCC95 and H226 cells with or without PD98 treatment cultured with the indicated conditioned medium derived from CrT/TICs. Data are presented as the mean ± SD of triplicate experiments. ** P < .001.

Article Snippet: DACH1 siRNA (sc‐77089), ABCB5 siRNA (sc‐89856), MERTK siRNA (sc‐37127), KLF4 siRNA (sc‐35480), SOX2 siRNA (sc‐38408), EGF siRNA (sc‐39416), and ALDH1A1 siRNA (sc‐41442) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Incubation, Cell Culture, Western Blot, Transfection, Derivative Assay, Recombinant

Journal: Clinical and Translational Medicine

Article Title: Chromium (VI)‐induced ALDH1A1/EGF axis promotes lung cancer progression

doi: 10.1002/ctm2.1136

Figure Lengend Snippet: ALDH1A1 inhibition increases the anti‐tumour effects of gemcitabine. (A) HCC95 or H226 cells (2 × 10 ⁶ ) mixed with or without CrT/TICs (1 × 10 ³ ) that stably expressed Dox‐inducible ALDH1A1 shRNA were orthotopically transplanted in the lungs of mice. After 5 days, mice were intraperitoneally injected with A37 or Dox triplicate times per week. Representative BLIs of orthotopic tumours and quantification of BLIs every 5 days are shown. (B) Kaplan–Meier survival curves for indicated mice. (C) IHC staining was performed with the indicated antibodies. Scale bar, 50 μm. (D) Tumour tissues were collected and homogenate. Immunoblot was conducted with indicated antibodies. (E) Representative TUNEL staining (green) and corresponding DAPI nuclear staining (blue) for indicated cells. (F) Drug treatment regimen for administration of A37 and gemcitabine (GEM). (G) HCC95 or H226 cells (2 × 10 ⁶ ) mixed with or without CrT/TICs (1 × 10 ³ ) were orthotopically transplanted in the lungs of mice. After 5 days, mice were treated with GEM. Representative BLIs of orthotopic tumours and quantification of BLIs every 14 days are shown. (H) Kaplan–Meier survival curves for indicated mice. MST, median survival time. (I) IHC staining was performed with anti‐Ki‐67. Scale bar, 50 μm. (J) Tumour tissues were collected and homogenate. Immunoblot was conducted with indicated antibodies. (K) Representative TUNEL staining (green) and corresponding DAPI nuclear staining (blue) for indicated cells

Article Snippet: DACH1 siRNA (sc‐77089), ABCB5 siRNA (sc‐89856), MERTK siRNA (sc‐37127), KLF4 siRNA (sc‐35480), SOX2 siRNA (sc‐38408), EGF siRNA (sc‐39416), and ALDH1A1 siRNA (sc‐41442) were purchased from Santa Cruz Biotechnology (CA, USA).

Techniques: Inhibition, Stable Transfection, shRNA, Injection, Immunohistochemistry, Western Blot, TUNEL Assay, Staining

Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: TNBC clinical samples express MerTK and the creation of a model of MerTK-overexpressing mouse TNBC cell lines. (A) MerTK is differentially expressed in human TNBC clinical samples as analyzed by IHC (n=137 patients). Pathological score was determined (D.T.Y) on a scale of 0-3+, and representative images for each score are shown. (B) MerTK is differentially expressed in human TNBC clinical samples as analyzed by IF (n=119 patients). Optical density (OD) was calculated using the inForm software. Representative images for each OD range are shown. (C) Whole cell lysate was harvested from 4T1 MerTK and EMT6 MerTK clones and their accompanying vector controls. MerTK expression was determined by immunoblot. GAPDH is shown as a loading control. (D) Cell culture supernatant was collected from confluent plates of 4T1 MerTK and EMT6 MerTK clones and their accompanying vector controls. Secreted Gas6 levels were analyzed by ELISA. (E) Lysates from 4T1 and EMT6 MerTK clones and their accompanying vector controls were evaluated for phosphorylation of downstream effectors of MerTK by phospho-protein array. (F) Pixel density of phosphorylated proteins circled in (E) . Mean values are shown (n=2). ** P<0.01; ns, not signficant.

Article Snippet: For the generation of MerTK tetracycline-inducible EMT6 clones, MerTK was subcloned into the pcDNA4/TO expression vector (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Software, Clone Assay, Plasmid Preparation, Expressing, Western Blot, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Protein Array

Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: MerTK expression promotes delayed tumor growth and a hotter TIME in syngeneic models of TNBC. (A) 4T1 MerTK and EMT6 MerTK overexpressing clones and their vector controls were injected onto the flanks of NCG mice, and tumor volume was measured twice weekly (n=8 tumors per group). (B) 4T1 MerTK and EMT6 MerTK overexpressing clones and their vector controls were injected onto the flanks of athymic nude mice, and tumor volume was measured twice weekly (n=8 tumors per group). (C) 4T1 MerTK and EMT6 MerTK overexpressing clones and their vector controls were injected onto the flanks of BALB/c mice, and tumor volume was measured twice weekly (n=6–16 tumors per group). Mice bearing EMT6 tumors were fed dox-containing chow (equivalent to 200mg/kg/day) beginning one week prior to tumor inoculation. Mean values and SEMs are shown. (D, E) Tumor-infiltrating lymphocytes (TILs) were analyzed via flow cytometry ( D : 4T1, E : EMT6). Mean values and SEMs are shown (n=5–10 tumors per group). (F) Cytokines and chemokines released by tumor cells into cell culture media were analyzed via Luminex. Mean values and SEMs are shown. (n = 3 per group). *P<0.05; ** P< 0.01; ***P<0.001; ns, not significant.

Article Snippet: For the generation of MerTK tetracycline-inducible EMT6 clones, MerTK was subcloned into the pcDNA4/TO expression vector (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Clone Assay, Plasmid Preparation, Injection, Flow Cytometry, Cell Culture, Luminex

Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: Inducing MerTK expression impairs tumor growth and promotes anti-tumor immune infiltration. (A) Vector and MerTK-overexpressing EMT6 cells were treated for 18 hours with vehicle or doxycycline (dox, 1ug/mL) prior to collecting whole-cell lysate. Lysates were immunoblotted for MerTK and GAPDH as a loading control. (B) Vector and MerTK-overexpressing EMT6 cells were inoculated onto the flanks of BALB/c mice. Tumor-bearing mice were treated with vehicle or doxycycline via chow (about 200mg/kg/day) starting at day 10 post-tumor inoculation, and tumor volume was measured twice weekly. Mean values and SEMs are shown (n=4–8 tumors per group). (C) Tumors were harvested and stained using IHC. Representative images are shown at 20x magnification. (D) Tumor immune infiltrate by IHC was quantified using FIJI V2.14.0/1.54f. Mean values and SEMs are shown (n=3–6 tumors per group). *P<0.05; ** P< 0.01; ***P<0.001; ns, not significant.

Article Snippet: For the generation of MerTK tetracycline-inducible EMT6 clones, MerTK was subcloned into the pcDNA4/TO expression vector (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Plasmid Preparation, Control, Staining

Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: Tumors overexpressing MerTK are sensitive to immune checkpoint inhibition. A) 4T1 tumors overexpressing MerTK were inoculated onto the flanks of BALB/c mice. Tumor-bearing mice were treated with either IgG, αPD-L1 (15mg/kg), or αCTLA-4 (5mg/kg) therapeutic antibody three to four times, three days apart. Mean values and SEMs are shown (n=14–18 tumors per group). (B) Spaghetti plots for tumors presented in (A) Red lines indicate subjects whose tumors regressed without recurrence. (C, D) Immune profiling of 4–10 tumors in each group that were harvested three days after the third dose of antibody. (C) Tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. Mean values and SEMs are shown (8–10 tumors per group). (D) Heatmap of significantly altered genes involved in anti-tumor immunity for IgG control, PD-L1, and CTLA-4 treatment groups, clustered by upregulated (green) or downregulated (purple). Red and blue represent upregulation and downregulation, respectively. (E) Tumors were analyzed for gene expression by NanoString nCounter. Mean values and SEMs are shown (4 tumors per group). * P <0.05; ** P < 0.01; *** P <0.001; ns, not significant.

Article Snippet: For the generation of MerTK tetracycline-inducible EMT6 clones, MerTK was subcloned into the pcDNA4/TO expression vector (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Inhibition, Flow Cytometry, Control, Gene Expression

Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: MerTK-negative tumors are resistant to immune checkpoint inhibition. (A) 4T1 vector and MerTK-overexpressing tumors were inoculated onto the flanks of BALB/c mice. Tumor-bearing mice were treated with IgG, αPD-L1 (15mg/kg), or αCTLA-4 (5mg/kg) therapeutic antibody four times 3–4 days apart. Mean values and SEMs are shown (n=12–16 tumors). (B) Spaghetti plots for each treatment group. Red lines indicate subjects whose tumors regressed without recurrence. (C) Cytotoxic granules (Granzyme B and Perforin), anti-tumor cytokines (IFN-γ and TNFα), and the apoptotic marker (CHOP/GADD153) were detected in tumor sections using IHC. Quantification was performed using FIJI V2.14.0/1.54f. Mean values and SEMs are shown (n=4–9 tumors per group). * P <0.05; ** P < 0.01; *** P <0.001; ns, not significant.

Article Snippet: For the generation of MerTK tetracycline-inducible EMT6 clones, MerTK was subcloned into the pcDNA4/TO expression vector (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Inhibition, Plasmid Preparation, Marker

Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: Human TNBCs expressing MerTK on the tumor exhibit increased lymphocyte recruitment. (A) Whole cell lysate was collected from SUM102 MerTK overexpressing clones and the vector control. Immunoblot analysis was performed for MerTK and GAPDH as a loading control. (B) MerTK expression was evaluated in SUM102 MerTK overexpressing clones and the vector control by flow cytometry. Fold changes relative to Vector control are shown (n=3 per group). (C) MerTK RNA expression was evaluated in SUM102 MerTK overexpressing clones and the vector control by qPCR. Fold changes relative to Vector control are shown (n=3 per group). (D) depiction of the KOALA assay workflow: (a) collection of human blood, (b) isolation of PBMCs, and (c) loading of PBMCs into lumens on the device base. (d) cultured cells were grown as spheroids and (e) embedded in the device lid. (f) The device lid is placed on the base, and the material flows through the lumen through capillary action. Created in BioRender. Wheeler, D. (2024) https://BioRender.com/f50n065 . (E) PBMC forward migration index (FMI) was evaluated for each spheroid type. Data points represent the horizontal center of mass, where negative FMI indicates the migration of PBMCs toward a stimulus. Mean values and SEMs are shown (n=33–35 cells per group). (F) Human clinical TNBC TMA samples were evaluated for tumor immune infiltration by multiplex IF. Representative images are shown. (G) Tumor immune infiltrate was quantified using inform software. Mean values and SEMs are shown (n=36–41 samples per group). ** P<0.01; *** p<0.001; ns, not significant.

Article Snippet: For the generation of MerTK tetracycline-inducible EMT6 clones, MerTK was subcloned into the pcDNA4/TO expression vector (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Clone Assay, Plasmid Preparation, Control, Western Blot, Flow Cytometry, RNA Expression, Isolation, Cell Culture, Migration, Multiplex Assay, Software

Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: Tumor-bound MerTK is not coupled to PD-L1 expression. (A) Whole cell lysate was collected from 4T1 cells overexpressing MerTK and the vector control and was immunoblotted for MerTK, PD-L1, and GAPDH, which was used as a loading control. (B) PD-L1 expression was analyzed in 4T1 cells overexpressing MerTK and vector control by flow cytometry. Mean values and SEMs are shown (n=3 per group). (C) PD-L1 RNA expression was evaluated in 4T1 cells overexpressing MerTK and vector control by NanoString nCounter. Mean Values and SEMs are shown (n=4 per group). (D) Correlation analysis of MerTK and PD-L1 mRNA expression in human TNBC patients. Data was obtained from the cBioportal (n=258 TNBC patients). (E) Correlation analysis of tumor-bound MerTK and stromal PD-L1 staining by mIF (n=101 TNBC patients). (F) Representative images of tumor-bound MerTK low, medium, and high-expressing patients. DAPI (blue), MerTK (cyan), and PD-L1 (magenta) staining are depicted for both stromal (green) and tumor (red) compartments. ns, not significant.

Article Snippet: For the generation of MerTK tetracycline-inducible EMT6 clones, MerTK was subcloned into the pcDNA4/TO expression vector (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Plasmid Preparation, Control, Flow Cytometry, RNA Expression, Staining

Journal: Frontiers in Oncology

Article Title: Tumor-associated MerTK promotes a pro-inflammatory microenvironment and enhances immune checkpoint inhibitor response in triple-negative breast cancer

doi: 10.3389/fonc.2025.1579214

Figure Lengend Snippet: High tumor-bound MerTK expression is associated with a hotter TIME and sensitivity to ICI in TNBC. Created in BioRender. Wheeler, D. (2024) https://BioRender.com/f41u920 .

Article Snippet: For the generation of MerTK tetracycline-inducible EMT6 clones, MerTK was subcloned into the pcDNA4/TO expression vector (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing

Journal: JCI Insight

Article Title: MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity

doi: 10.1172/jci.insight.97941

Figure Lengend Snippet: 697 B-ALL cells expressing the firefly luciferase gene were inoculated into NSG mice by tail vein injection. Disease burden was assessed by bioluminescence imaging, and survival was monitored. (A–D) Mice were treated with 50 mg/kg (dashed green line) or 75 mg/kg (solid green line) MERTK inhibitor MRX-2843 or an equivalent volume of vehicle (saline; solid black line) once daily, beginning 1 day after leukemia cell injection to model low leukemic burden. (B) Representative bioluminescence images. (C) Mean bioluminescence intensities and SEM (n = 10–11, *P < 0.05, **P < 0.01, ****P < 0.0001, ‡P < 0.05, ‡‡‡‡P <0.0001; 1-way ANOVA; *comparison between saline and 75 mg/kg UNC2025); ‡comparison between saline and 50 mg/kg UNC2025. (D) Kaplan-Meier survival curves (n = 7–10, P < 0.0001 compared with vehicle, log-rank test). (E–H) Leukemic mice were randomized to groups with equal mean bioluminescence intensities and treated with 75 mg/kg MRX-2843 or an equivalent volume of vehicle (saline) daily, beginning 12 days after tumor inoculation to model existent disease. (F) Representative bioluminescence images. (G) Mean bioluminescence intensities and standard errors (n = 10). (H) Kaplan-Meier survival curves (n = 10, P < 0.0001 compared with vehicle, log-rank test). Data shown are representative of 2 independent experiments.

Article Snippet: The following antibodies were used to assess in vivo coinhibitory receptor expression: anti–mouse CD11b–APC-CY7 (clone M1/70) and anti-mouse CD3-PE (clone 17A2) from BD Biosciences; goat anti–mouse MERTK (polyclonal) from R&D Systems; anti–mouse CD279–BV421 (PD-1, clone 29F1A12), anti–mouse CD274–BV421 (PD-L1, clone 10F9G2), and anti–mouse CD273–PE (PD-L2, clone TY25) from BioLegend; anti–mouse CD4–PE-Cy7 (clone RM4-5) and anti–mouse CD8α–APC-eFluor780 (clone 53-6.7) from eBioscience.

Techniques: Expressing, Luciferase, Injection, Imaging, Saline, Comparison

Journal: JCI Insight

Article Title: MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity

doi: 10.1172/jci.insight.97941

Figure Lengend Snippet: WT or Mertk–/– C57BL/6 mice were inoculated with 5 × 104 GFP-expressing Arf–/– p185+ B-ALL cells by tail vein injection. (A) MERTK expression was not detected in the Arf–/– BCR-ABL p185+ B-ALL cell line by immunoblot. The J774 murine macrophage-like cell line is shown as a positive control. (B) Kaplan-Meier survival curves derived from WT or Mertk–/– C57BL/6 mice injected with B-ALL cells (WT n = 30 and Mertk–/– n = 20 in 3 independent cohorts). (C) Kaplan-Meier survival curves derived from WT mice injected with B-ALL cells and treated with 10 mg/kg MERTK inhibitor MRX-2843 twice daily (BID), 60 mg/kg MRX-2843 once daily, or an equivalent volume of vehicle (saline) beginning 1 day after transplant (n = 13 in 2 independent cohorts). (D) Kaplan-Meier survival curve derived from WT mice injected with B-ALL cells and treated with 60 mg/kg MRX-2843 once daily starting the day after injection (Immediate MRX-2843) or 5 days after injection (Delayed start MRX-2843), or an equivalent volume of vehicle (saline) beginning 1 day after transplant (n = 8, representative of 2 independent cohorts). (E) Kaplan-Meier survival curve derived from NSG mice injected with 5,000 Arf–/– p185+ cells (n = 6–10, representative of 2 independent experiments). Statistically significant differences were determined compared with WT or vehicle using a log-rank test.

Article Snippet: The following antibodies were used to assess in vivo coinhibitory receptor expression: anti–mouse CD11b–APC-CY7 (clone M1/70) and anti-mouse CD3-PE (clone 17A2) from BD Biosciences; goat anti–mouse MERTK (polyclonal) from R&D Systems; anti–mouse CD279–BV421 (PD-1, clone 29F1A12), anti–mouse CD274–BV421 (PD-L1, clone 10F9G2), and anti–mouse CD273–PE (PD-L2, clone TY25) from BioLegend; anti–mouse CD4–PE-Cy7 (clone RM4-5) and anti–mouse CD8α–APC-eFluor780 (clone 53-6.7) from eBioscience.

Techniques: Expressing, Injection, Western Blot, Positive Control, Derivative Assay, Saline

Journal: JCI Insight

Article Title: MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity

doi: 10.1172/jci.insight.97941

Figure Lengend Snippet: C57BL/6 WT or Mertk–/– mice were injected intravenously with Arf–/– p185+ B-ALL cells or saline. When symptoms of leukemia were evident in at least 1 mouse, bone marrow and spleens were harvested from those animals and their cagemates (1 per group per cage) and stained for flow cytometric analysis. (A) Representative flow cytometry profiles showing PD-L1 and PD-L2 expression on GFP– CD11b+ myeloid cells isolated from the spleens of WT and Mertk–/– mice with and without inoculation of B-ALL. (B) Percentages of PD-L1+ PD-L2+ cells within the GFP– CD11b+ population in bone marrow and spleen. (C and D) WT mice were treated with 10 mg/kg MERTK inhibitor (MRX-2843) twice daily (BID), 60 mg/kg MRX-2843 once daily, or vehicle (saline) starting 1 day after inoculation. (C) Representative flow cytometry profiles showing PD-L1 and PD-L2 expression on GFP– CD11b+ myeloid cells isolated from the spleen. (D) Percentages of PD-L1+ PD-L2+ cells within the GFP– CD11b+ population in bone marrow and spleen. Mean values and standard errors from two independent cohorts are shown. (n = 3–11, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; 1-way ANOVA).

Article Snippet: The following antibodies were used to assess in vivo coinhibitory receptor expression: anti–mouse CD11b–APC-CY7 (clone M1/70) and anti-mouse CD3-PE (clone 17A2) from BD Biosciences; goat anti–mouse MERTK (polyclonal) from R&D Systems; anti–mouse CD279–BV421 (PD-1, clone 29F1A12), anti–mouse CD274–BV421 (PD-L1, clone 10F9G2), and anti–mouse CD273–PE (PD-L2, clone TY25) from BioLegend; anti–mouse CD4–PE-Cy7 (clone RM4-5) and anti–mouse CD8α–APC-eFluor780 (clone 53-6.7) from eBioscience.

Techniques: Injection, Saline, Staining, Flow Cytometry, Expressing, Isolation

Journal: JCI Insight

Article Title: MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity

doi: 10.1172/jci.insight.97941

Figure Lengend Snippet: Splenocytes harvested from WT or Mertk–/– mice were cultured at a 1:1 ratio with Arf–/– p185+ B-ALL cells and treated with 200 nM MRX-2843 or vehicle (DMSO) for 72 hours. The indicated cell surface proteins were quantitated by flow cytometry. (A) Representative dot plots showing PD-L1+ PD-L2+ cells within the GFP–CD11b+ population. (B) Graphical representation of the percentage of GFP–CD11b+ cells coexpressing PD-L1+ and PD-L2+. Mean values and standard errors from 2 independent cohorts are shown. (****P < 0.0001; 2-way ANOVA).

Article Snippet: The following antibodies were used to assess in vivo coinhibitory receptor expression: anti–mouse CD11b–APC-CY7 (clone M1/70) and anti-mouse CD3-PE (clone 17A2) from BD Biosciences; goat anti–mouse MERTK (polyclonal) from R&D Systems; anti–mouse CD279–BV421 (PD-1, clone 29F1A12), anti–mouse CD274–BV421 (PD-L1, clone 10F9G2), and anti–mouse CD273–PE (PD-L2, clone TY25) from BioLegend; anti–mouse CD4–PE-Cy7 (clone RM4-5) and anti–mouse CD8α–APC-eFluor780 (clone 53-6.7) from eBioscience.

Techniques: Cell Culture, Flow Cytometry

Journal: JCI Insight

Article Title: MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity

doi: 10.1172/jci.insight.97941

Figure Lengend Snippet: C57BL/6 WT or Mertk–/– mice were injected intravenously with Arf–/– p185+ B-ALL cells or saline, and bone marrow and spleens were harvested and stained for flow cytometric analysis as described in Figure 3. (A) Representative flow cytometry profiles showing PD-1 expression on CD8+ T cells within the GFP–CD3+ population in spleens of WT and Mertk–/– mice. (B) Percentages of PD-1+ cells within the GFP–CD3+ CD4+ and CD8+ populations in spleens of WT and Mertk–/– mice. (C) Representative flow cytometry profiles showing PD-1 expression on CD8+ T cells within the GFP–CD3+ population isolated from the spleens of WT mice treated with MRX-2843 or vehicle (saline). (D) Percentages of PD-1+ cells within the GFP–CD3+ CD4+ and CD8+ population in spleens of WT mice treated with MRX-2843. Mean values and standard errors from 2 independent cohorts are shown. (n = 3–12). ***P < 0.001, ****P < 0.0001; 1-way ANOVA).

Article Snippet: The following antibodies were used to assess in vivo coinhibitory receptor expression: anti–mouse CD11b–APC-CY7 (clone M1/70) and anti-mouse CD3-PE (clone 17A2) from BD Biosciences; goat anti–mouse MERTK (polyclonal) from R&D Systems; anti–mouse CD279–BV421 (PD-1, clone 29F1A12), anti–mouse CD274–BV421 (PD-L1, clone 10F9G2), and anti–mouse CD273–PE (PD-L2, clone TY25) from BioLegend; anti–mouse CD4–PE-Cy7 (clone RM4-5) and anti–mouse CD8α–APC-eFluor780 (clone 53-6.7) from eBioscience.

Techniques: Injection, Saline, Staining, Flow Cytometry, Expressing, Isolation

Journal: JCI Insight

Article Title: MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity

doi: 10.1172/jci.insight.97941

Figure Lengend Snippet: CD4+ and CD8+ T cells were isolated from spleens from C57BL/6 WT or Mertk–/– mice and stimulated for 24 hours with anti-CD3 antibody, then cultured at a 2:1:1 ratio with Arf–/– p185+ B-ALL cells and WT or Mertk–/– splenocytes and treated with 200 nM MRX-2843 or vehicle (DMSO) for 72 hours. Production of IFN-γ and TNF-α in CD4+ and CD8+ T cells was measured by flow cytometry. (A and C) Representative dot plots showing IFN-γ and TNF-α levels in CD4+ (A) and CD8+ (C) T cells. (B and D) Graphical representation of the percentage of CD4+ and CD8+ T cells expressing both IFN-γ and TNF-α. Mean values and standard errors from 3 independent experiments are shown. ***P < 0.001, ****P < 0.0001; 2-way ANOVA.

Article Snippet: The following antibodies were used to assess in vivo coinhibitory receptor expression: anti–mouse CD11b–APC-CY7 (clone M1/70) and anti-mouse CD3-PE (clone 17A2) from BD Biosciences; goat anti–mouse MERTK (polyclonal) from R&D Systems; anti–mouse CD279–BV421 (PD-1, clone 29F1A12), anti–mouse CD274–BV421 (PD-L1, clone 10F9G2), and anti–mouse CD273–PE (PD-L2, clone TY25) from BioLegend; anti–mouse CD4–PE-Cy7 (clone RM4-5) and anti–mouse CD8α–APC-eFluor780 (clone 53-6.7) from eBioscience.

Techniques: Isolation, Cell Culture, Flow Cytometry, Expressing

Journal: JCI Insight

Article Title: MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity

doi: 10.1172/jci.insight.97941

Figure Lengend Snippet: C57BL/6 WT or Mertk–/– mice were injected intravenously with Arf–/– p185+ B-ALL cells or saline, and bone marrow and spleens were harvested and stained for flow cytometric analysis as described in Figure 3. (A) Representative flow cytometry profiles showing CD4+ FOXP3+ Tregs within the GFP– CD3+ population isolated from the spleens of WT and Mertk–/– mice. (B) Percentages of CD4+ FOXP3+ cells within the GFP–CD3+ population in bone marrow and spleens of WT and Mertk–/– mice. (C) Representative flow cytometry profiles identifying the CD4+FOXP3+ T regulatory cells within the GFP– CD3+ population isolated from the spleens of WT mice treated with MERTK inhibitor MRX-2843 or vehicle (saline). (D) Percentages of CD4+FOXP3+ cells within the GFP–CD3+ population in bone marrow and spleens of WT mice treated with MRX-2843. Mean values and standard errors from 2 independent cohorts are shown. (n = 3–12, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; 1-way ANOVA).

Article Snippet: The following antibodies were used to assess in vivo coinhibitory receptor expression: anti–mouse CD11b–APC-CY7 (clone M1/70) and anti-mouse CD3-PE (clone 17A2) from BD Biosciences; goat anti–mouse MERTK (polyclonal) from R&D Systems; anti–mouse CD279–BV421 (PD-1, clone 29F1A12), anti–mouse CD274–BV421 (PD-L1, clone 10F9G2), and anti–mouse CD273–PE (PD-L2, clone TY25) from BioLegend; anti–mouse CD4–PE-Cy7 (clone RM4-5) and anti–mouse CD8α–APC-eFluor780 (clone 53-6.7) from eBioscience.

Techniques: Injection, Saline, Staining, Flow Cytometry, Isolation

Journal: JCI Insight

Article Title: MERTK inhibition alters the PD-1 axis and promotes anti-leukemia immunity

doi: 10.1172/jci.insight.97941

Figure Lengend Snippet: Model depicting roles for MERTK in innate immune responses in the leukemia microenvironment. (A) In the presence of ALL, MERTK induces coexpression of the coinhibitory ligands PD-L1 and PD-L2 on CD11b+ monocytes/macrophages, and indirectly induces expression of the coinhibitory receptor PD-1 on CD8+ T cells and promotes an increased incidence of Tregs. (B) In response to MERTK inhibition, coexpression of PD-L1 and PD-L2 decreases on CD11b+ cells, expression of PD-1 on MERTK-negative T cells decreases, and regulatory T cells are reduced, leading to T cell activation and induction of anti-leukemia immunity and ultimately resulting in reduced leukemic burden. TCR, T cell receptor.

Article Snippet: The following antibodies were used to assess in vivo coinhibitory receptor expression: anti–mouse CD11b–APC-CY7 (clone M1/70) and anti-mouse CD3-PE (clone 17A2) from BD Biosciences; goat anti–mouse MERTK (polyclonal) from R&D Systems; anti–mouse CD279–BV421 (PD-1, clone 29F1A12), anti–mouse CD274–BV421 (PD-L1, clone 10F9G2), and anti–mouse CD273–PE (PD-L2, clone TY25) from BioLegend; anti–mouse CD4–PE-Cy7 (clone RM4-5) and anti–mouse CD8α–APC-eFluor780 (clone 53-6.7) from eBioscience.

Techniques: Expressing, Inhibition, Activation Assay

Journal: Redox Biology

Article Title: Endothelial MerTK impairment accelerates the development of atherosclerosis

doi: 10.1016/j.redox.2025.103861

Figure Lengend Snippet: Big data analytics and human microarray reveal the key signaling pathways in atherosclerosis. Big data analytics. ( A – C ) Big data analytics for atherosclerosis with 98881cross analyses for overall signaling and 234 cross analyses in aortic arch based on up-to-date RNA-seq data from humans, mouse and rat. In IPA of Pathways and Lists, atherosclerosis was set as the keywords. Microarray in human atherosclerosis. ( D – E ) The top 50 downregulated or upregulated upstream regulators based on activation of z-score. ( F ) Graphical summary of human microarray data (orange: upregulated; blue: downregulated). QIAGEN Ingenuity Pathway Analysis (IPA: 1-atherosclerosis [carotid atherosclerotic plaque] NA CMP_2gGgljQ5SpJAn) and QIAGEN OmicSoft Land Explorer (OLE) were used to analyze microarray data in carotid atherosclerotic plaque from human patients. RNA-seq big data analytics in human . ( G ) MerTK expression in human diseases specifically in related aortic tissues (n = 427 in total), including normal control, embryo, cardiovascular disease (CVD) and respiratory tract disease (RTD)-related cardiovascular disease. MerTK expression was based on RNA-seq or scRNA-seq and was quantified by Log2 (FPKM + 0.1). Original data of RNA-seq or scRNA-seq for MerTK expression were downloaded from QIAGEN OmicSoft Land Explorer. BioGPS . ( H ) MerTK mRNA expression in human cells derived from BioGPS ( http://biogps.org ). The data were analyzed with GraphPad Prism 9.4.1 and shown as the mean ± SD.

Article Snippet: MerTK flox/flox Tie2 Cre mice with MerTK conditional knockout in ECs were generated by crossing MerTK flox/flox mice with Tie2-Cre mice ( Cyagen US, Santa Clara, CA ) .

Techniques: Microarray, Protein-Protein interactions, RNA Sequencing, Activation Assay, Expressing, Control, Derivative Assay

Journal: Redox Biology

Article Title: Endothelial MerTK impairment accelerates the development of atherosclerosis

doi: 10.1016/j.redox.2025.103861

Figure Lengend Snippet: Endothelial MerTK deficiency promotes the development of atherosclerosis. ( A ) Body weight measurement in MerTK flox/flox mice and MerTK flox/flox Tie2 Cre mice of atherosclerosis model. ( B – C ) The visible formation of atherosclerotic plaque and H&E staining in aortic arch. After the mice were euthanized, the whole aortas were carefully dissected from surrounding tissue and fixed with 10 % neutral buffered formalin solution. The visible formation of atherosclerotic plaque was acquired by an AmScope Trinocular Stereo Microscope with a 12 MP digital camera. ( D ) Immunostaining for Cav-1 expression in aortic arch from MerTK flox/flox mice and MerTK flox/flox Tie2 Cre mice of atherosclerosis model. Mice were injected with a single dose of AAV8-PCSK9 particles along with a high fat diet for two months.

Article Snippet: MerTK flox/flox Tie2 Cre mice with MerTK conditional knockout in ECs were generated by crossing MerTK flox/flox mice with Tie2-Cre mice ( Cyagen US, Santa Clara, CA ) .

Techniques: Staining, Microscopy, Immunostaining, Expressing, Injection

Journal: Redox Biology

Article Title: Endothelial MerTK impairment accelerates the development of atherosclerosis

doi: 10.1016/j.redox.2025.103861

Figure Lengend Snippet: Endothelial MerTK deficiency promotes proinflammation response and activates p22 phox while inhibits ApoE expression. ( A – D ) Immunostaining for the expression of IL-1β, MCP-1, ApoE, p22 phox and NF-κB i n aortic arch from MerTK flox/flox mice and MerTK flox/flox Tie2 Cre mice that were injected with a single dose of AAV8-PCSK9 particles along with a high fat diet for two months.

Article Snippet: MerTK flox/flox Tie2 Cre mice with MerTK conditional knockout in ECs were generated by crossing MerTK flox/flox mice with Tie2-Cre mice ( Cyagen US, Santa Clara, CA ) .

Techniques: Expressing, Immunostaining, Injection

Journal: Redox Biology

Article Title: Endothelial MerTK impairment accelerates the development of atherosclerosis

doi: 10.1016/j.redox.2025.103861

Figure Lengend Snippet: Endothelial MerTK deficiency activates pro-atherosclerotic factors. ( A – D ) Immunostaining for the expression of inflammation markers (TNF-α and IFN-γ), NADPH oxidase subunits (p47 phox and gp91 phox ), and MAPK family (ERK, p38 and JNK) in aortic arch from MerTK flox/flox mice and MerTK flox/flox Tie2 Cre mice that were injected with a single dose of AAV8-PCSK9 particles along with a high fat diet for two months.

Article Snippet: MerTK flox/flox Tie2 Cre mice with MerTK conditional knockout in ECs were generated by crossing MerTK flox/flox mice with Tie2-Cre mice ( Cyagen US, Santa Clara, CA ) .

Techniques: Immunostaining, Expressing, Injection

Journal: Redox Biology

Article Title: Endothelial MerTK impairment accelerates the development of atherosclerosis

doi: 10.1016/j.redox.2025.103861

Figure Lengend Snippet: Proteomics shows the key signaling pathways in endothelial MerTK-mediated atherosclerosis . ( A ) Protein abundance in comparison of MerTK flox/flox group with MerTK flox/flox Tie2 Cre group. ( B ) Volcano plot illustrating differentially expressed proteins in the aortic arch of MerTK flox/flox Tie2 Cre vs. MerTK flox/flox . Relative protein abundance (log2) plotted against significance level (-log10 P-value), showing downregulated (blue), upregulated (red) or non-differentially expressed proteins (grey). ( C ) Graphical summary of proteomics data (orange: upregulated; blue: downregulated). ( D ) The volcano canonical pathways based on activation of z-score (lower panel). Blue: negative value. Orange: positive value. Grey: no activity pattern. Size is based on the number of genes that overlap the pathway. Big data analytics for 67,629 cross analyses for mitochondrial dysfunction based on IPA data base (upper panel). ( E – F ) The top 50 downregulated or upregulated upstream regulators based on activation of z-score.

Article Snippet: MerTK flox/flox Tie2 Cre mice with MerTK conditional knockout in ECs were generated by crossing MerTK flox/flox mice with Tie2-Cre mice ( Cyagen US, Santa Clara, CA ) .

Techniques: Protein-Protein interactions, Quantitative Proteomics, Comparison, Activation Assay, Activity Assay

Journal: Redox Biology

Article Title: Endothelial MerTK impairment accelerates the development of atherosclerosis

doi: 10.1016/j.redox.2025.103861

Figure Lengend Snippet: MerTK flox/flox Tie2 Cre group demonstrates aggravated mitochondrial dysfunction compared to MerTK flox/flox group. ( A - B ) Machine learning (ML) disease pathways showing mitochondrial dysfunctions and the protein changes of mitochondrial DNA-related disorder (orange: upregulated; blue: downregulated). ( C – F ) Graphical summary for mitochondrial DNA-related disorder, mitochondrial disorder, mitochondrial cytopathy, and mitochondrial myopathy. Red: increased measurement. Green: decreased measurement. Orange: predicted activation. Bule: predicted inhibition. Glos indicates activity when the opposite of measurement. The lines indicate the predicated relationship (orange: leads to activation; blue: leads to inhibition; yellow: findings inconsistent with state of downstream molecule; grey: effect not predicted). ( G – H ) Activated or inhibited molecules associated with mitochondrial dysfunction.

Article Snippet: MerTK flox/flox Tie2 Cre mice with MerTK conditional knockout in ECs were generated by crossing MerTK flox/flox mice with Tie2-Cre mice ( Cyagen US, Santa Clara, CA ) .

Techniques: Activation Assay, Inhibition, Activity Assay

Journal: Redox Biology

Article Title: Endothelial MerTK impairment accelerates the development of atherosclerosis

doi: 10.1016/j.redox.2025.103861

Figure Lengend Snippet: Proteomics for causal network analysis shows top 50 changed regulators and microRNAs in aortic arch with MerTK flox/flox Tie2 Cre vs. MerTK flox/flox . ( A ) The top 50 activated upstream regulators and IPA prediction of MAPK family and TGFβ family networks based on activation of z-score. ( B ) The top 50 inhibited upstream regulators and IPA prediction of MAPK inhibitor and SB203580 networks based on activation of z-score. ( C ) The upregulated or downregulated microRNAs in MerTK flox/flox Tie2 Cre group compared to MerTK flox/flox group. Representative IPA prediction micorRNA-218 focusing on MAPK signaling pathway. Upregulated and downregulated proteins are highlighted in red and green, respectively, and the color depth is correlated to the fold change. Orange and blue dashed lines with arrows indicate indirect activation and inhibition, respectively. Yellow and grey dashed lines with arrows depict inconsistent effects and no prediction, respectively.

Article Snippet: MerTK flox/flox Tie2 Cre mice with MerTK conditional knockout in ECs were generated by crossing MerTK flox/flox mice with Tie2-Cre mice ( Cyagen US, Santa Clara, CA ) .

Techniques: Activation Assay, Inhibition

Journal: Redox Biology

Article Title: Endothelial MerTK impairment accelerates the development of atherosclerosis

doi: 10.1016/j.redox.2025.103861

Figure Lengend Snippet: Multi-comparisons for common signaling between proteomics of MerTK flox/flox Tie2 Cre vs. MerTK flox/flox and shared atherosclerosis projects. ( A ) Upstream regulators in MerTK flox/flox Tie2 Cre vs. MerTK flox/flox compared with other atherosclerosis projects based on activation of z-score. ( B ) The volcano canonical pathways based on activation of z-score. ( C ) The toxicity functions analyses based on activation of z-score. ( D ) Diseases and biological functions analysis based on activation of z-score.

Article Snippet: MerTK flox/flox Tie2 Cre mice with MerTK conditional knockout in ECs were generated by crossing MerTK flox/flox mice with Tie2-Cre mice ( Cyagen US, Santa Clara, CA ) .

Techniques: Activation Assay

Journal: Redox Biology

Article Title: Endothelial MerTK impairment accelerates the development of atherosclerosis

doi: 10.1016/j.redox.2025.103861

Figure Lengend Snippet: The axis of miR-218-5p/EC MerTK /MAPK represents a novel mechanism of endothelial MerTK-mediated atherosclerosis. ( A ) Expression of p-JNK, p-p38 and p-ERK in HAECs ( B ) Immunostaining for p-ERK expression in HAECs. ( A - B ) HAECs were transfected with transfected with MerTK CRISPR/Cas9 KO Plasmid or control plasmid at 1 μg/6-well in 1 mL for 48 h. Then HAECs were incubated with apoptotic Jurkat cells at a 1:1 apoptotic cell/EC ratio. ( C ) Expression of p-JNK, p-p38 and p-ERK in HAECs. Cells were pre-transfected with miR-218–5p or miR control at 100 nM for 48 h, treated with MerTK inhibitors of UNC 569 or UNC 5293 at 500 nM for 4 h, and then were incubated with apoptotic cells at a 1:1 apoptotic cell/EC ratio for 1 h.

Article Snippet: MerTK flox/flox Tie2 Cre mice with MerTK conditional knockout in ECs were generated by crossing MerTK flox/flox mice with Tie2-Cre mice ( Cyagen US, Santa Clara, CA ) .

Techniques: Expressing, Immunostaining, Transfection, CRISPR, Plasmid Preparation, Control, Incubation

Journal: Cell reports

Article Title: Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses

doi: 10.1016/j.celrep.2022.110600

Figure Lengend Snippet: (A) Representative confocal image of microglia (green; CX3CR1-GFP) abutting CRH + neurons (red; CRH-tdTomato), engulfing vGlut2 + excitatory presynaptic puncta (white; vGlut2 puncta identified as inside microglia by Imaris 3D reconstruction) in mpd PVN of a P8 male mouse. Scale bar, 10 μm (raw images in – ). (B) Representative electron micrograph of a microglia labeled with ionized calcium binding adaptor molecule (Iba)1 and 3′,3-diaminobenzidine (DAB; electron-dense soma and processes) in P8 PVN. The yellow arrowhead points to the postsynaptic density of an excitatory synapse directly abutting a labeled microglial process. The black arrowhead indicates multiple lysosomes (electron dense) in microglial soma; these degrade engulfed material. The blue arrowhead points to an engulfed endosome of synaptic elements, including a putative postsynaptic density, surrounded by lysosomes in a microglia process. Scale bar, 5 μm. (C) ELA reduces the number of vGlut2 + synaptic puncta engulfed by microglia abutting CRH + neurons in the P8 mpd PVN of male mice (t 13.87 = 2.22, p = 0.04; Welch’s t test; see for female data), as quantified in confocal 3D reconstructions. (D) Representative confocal image of the microglial phagocytic receptor Mer tyrosine kinase (MerTK) expression. MerTK is primarily in microglia in the P8 PVN (white ROI; see also ). Red = CRH-tdTomato + neurons; green = MerTK immunoreactivity (IR); magenta = P2RY12 IR (microglia); white = overlap of green and magenta in composite image. Scale bar, 50 μm. (E) Representative confocal images of MerTK IR in CTL (top) compared to ELA (bottom) P8 PVN (white ROI). Scale bar, 50 μm. (F) MerTK volume per unit volume of P2RY12 + microglia (measured using Imaris) was lower in P8 ELA male PVN than in controls (t 6.46 = 3.44, p = 0.01; Welch’s t test). (G) Notably, P2RY12 volume and its percentage of PVN volume did not differ in ELA compared to control mice. (H) The ratio of MerTK mean fluorescence intensity (MFI; measured using Imaris) to P2RY12 MFI was significantly reduced in ELA mice PVN (t 9 = 2.54, p = 0.03; unpaired t test). (I) Schematic of the MerTK inhibition experiment: Litters of CRH-tdTomato + mice were randomly assigned to CTL or ELA conditions on P2. On P6–7, organotypic hypothalamic slice cultures were prepared and maintained for 7 d in vitro (DIV). On DIV7, cultures were treated with 20 nM of a small-molecule MerTK inhibitor (UNC2025: ~40-fold greater selectivity for MerTK over the Axl and Tyro3 TAM receptors [ ; ]) or vehicle. Twelve hours later, the medium was refreshed with new drug, and the cultures were fixed 4 h later . (J) MerTK inhibition increased the number of excitatory synapses on CRH + neurons in PVN cultures from control mice but not in ELA mice (significant interaction of ELA × drug, F 1,22 = 17.89, p = 0.0003; 2-way ANOVA; p < 0.05; post hoc Tukey’s test). Means ± SEMs; *p < 0.05.

Article Snippet: 6 days after initial culture, each membrane was washed twice with 2 mL of antibiotic-free, serum-free medium (97% MEM supplemented with 3 mM L-Glutamine, 10 mM D-Glucose,1.9 mM NaHCO3, 12.5 mM HEPES, 0.6 mM L-Ascorbic acid, 1 μg/mL Insulin) and treatment with 20 nM of a small-molecule MerTK inhibitor as previously described (UNC2025, Selleck Chemicals, cat# S7576) ( ) or sterile culture-grade water as vehicle was commenced.

Techniques: Labeling, Binding Assay, Expressing, Control, Fluorescence, Inhibition, In Vitro

Journal: Cell reports

Article Title: Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses

doi: 10.1016/j.celrep.2022.110600

Figure Lengend Snippet:

Article Snippet: 6 days after initial culture, each membrane was washed twice with 2 mL of antibiotic-free, serum-free medium (97% MEM supplemented with 3 mM L-Glutamine, 10 mM D-Glucose,1.9 mM NaHCO3, 12.5 mM HEPES, 0.6 mM L-Ascorbic acid, 1 μg/mL Insulin) and treatment with 20 nM of a small-molecule MerTK inhibitor as previously described (UNC2025, Selleck Chemicals, cat# S7576) ( ) or sterile culture-grade water as vehicle was commenced.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Transgenic Assay, Software, Microscopy, Imaging

Journal: Scientific Reports

Article Title: Glucocorticoids induce a phagocytic C1Q+ macrophage phenotype primed for IFNγ-dependent CXCL9 secretion

doi: 10.1038/s41598-026-52733-y

Figure Lengend Snippet: GC-induced MΦ are highly efficient in C1q-mediated phagocytosis. ( A ) Gene set enrichment analysis (GSEA) of DEGs in MΦ exposed to CM of steroidogenic NCI-H295R cells in comparison to controls. ( B ) Visualization of the enrichment results on phagocytosis ( p < 2.2e-16; FDR: 0.011718). ( C ) Representative flow cytometry plots and ( D ) quantification of phagocytosis by MΦ exposed to CM of steroidogenic NCI-H295R cells or control. CD64 + CFSE+ double-positive cells were considered phagocytic macrophages. Groups were compared using one-way ANOVA with Tukey’s multiple comparisons test. N = 4 biological replicates from independent healthy blood donors ( E ) Percentages of phagocytic MΦ exposed to dexamethasone (100 nM) or control. N = 3 biological replicates from independent healthy blood donors are shown in mean +/- SD. Differences were compared using unpaired t-test. ( F ) Phagocytosis of M1-polarized MΦ after exposure to 10 µg/ml or 20 µg/ml purified human C1q. N = 3 biological replicates from independent healthy blood donors are shown in mean +/- SD. Groups were compared using one-way ANOVA with Tukey’s multiple comparisons test. ( G ) Phagocytosis of dexamethasone (100 nM) -polarized MΦ after exposure to 1 µg/ml, 10 µg/ml or 50 µg/ml anti-C1QA antibody. N = 3 biological replicates from independent healthy blood donors are shown in mean +/- SD. Groups were compared using one-way ANOVA with Tukey’s multiple comparisons test. ( H ) Phagocytosis of dexamethasone (100 nM)-polarized MΦ after exposure to the MerTK inhibitor UNC1062 (100 nM), anti-Gas6 antibody (20 µg/ml) or goat control IgG (20 µg/ml). N = 3 biological replicates from independent healthy blood donors are shown in mean +/- SD. Groups were compared using one-way ANOVA with Tukey’s multiple comparisons test. ( I ) Kaplan-Meier plots of PFS and OS according to tumoral CSF1 and C1QA expression. Higher gene expression was significantly associated with favorable PFS and OS. RNA expression and survival data was extracted from the TCGA dataset ( n = 76). NCI-R = NCI-H295R; mety=metyrapone.

Article Snippet: For inhibition studies, MФ were pre-incubated with the selective MerTK inhibitor UNC1062 (100 nM; MCE: Cat# HY-117548), anti-Gas6 antibody (20 μg/ml; R&D Systems, Cat# AB885, RRID: AB_354376), a goat isotype IgG control (20 μg/ml; Thermo Fisher Scientific, Cat# 02–6202, RRID: AB_2532946), purified C1q (Merck; Cat# C1740) or anti-C1QA antibody (LS-Bio; Cat# LS‐C100911; RRID: AB_2067267) for 30 min before they were added to early ferroptotic tumor cells.

Techniques: Comparison, Flow Cytometry, Control, Purification, Expressing, Gene Expression, RNA Expression

Journal: Frontiers in Immunology

Article Title: Induction of hepatic fibrosis in mice with schistosomiasis by extracellular microRNA-30 derived from Schistosoma japonicum eggs

doi: 10.3389/fimmu.2024.1425384

Figure Lengend Snippet: miRNA-30 derived from S. japonicum egg exosomes activates HSCs. (A) Exosomes were observed under transmission electron microscopy (TEM). (B) LX-2 cells were transfected with miRNA mimics for 48 h, followed by qPCR analysis to measure the mRNA levels of α-SMA . (C) LX-2 and mHSC cells were transfected with 50 nM of both NC mimic and miRNA-30 mimic, for 24 (h) Relative transcript levels of miRNA-30 were determined by qPCR. (D) LX-2 and mHSC cells were transfected with varying doses of NC mimic and miR-30 mimic for 48 h, followed by qPCR to quantify the mRNA levels of α-SMA , Col1(α1) , and Col3(α1) . (E) LX-2 cells were exposed to a 100 nM of both NC mimic and miRNA-30 mimic for 48 (h) The LX-2 cells were then subjected to Western blot analysis to detect their α-SMA protein expression levels. Both mRNA and protein levels of α-SMA , Col1(α1) , and Col3(α1) were normalized against GAPDH . GAPDH , 37 kDa; α-SMA , 43 kDa. NC, negative control; 30 mimic, miRNA-30 mimic. The results are averaged from three independent experiments. Student’s t -tests were carried out to assess differences between the two groups (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Article Snippet: The membrane was then incubated with rabbit anti-GAPDH (5174S, CST, USA), anti-Col1(α1) (bs-10423R, Bioss Antibodies, USA), anti-Col3(α1) (bs-0548R, Bioss Antibodies), or anti-α-SMA (19245S, CST, USA) antibodies overnight at 4°C, followed by three 1×TBST washes.

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Transfection, Western Blot, Expressing, Negative Control

Journal: Frontiers in Immunology

Article Title: Induction of hepatic fibrosis in mice with schistosomiasis by extracellular microRNA-30 derived from Schistosoma japonicum eggs

doi: 10.3389/fimmu.2024.1425384

Figure Lengend Snippet: Overexpression of miRNA-30 induces hepatic fibrosis in naive mice. Healthy six-week-old female C57BL/6 mice were injected with of PBS, rAAV8-SCR (blank vector), and rAAV8-miR-30 (overexpression vector) into the tail vein, with a viral titer of 5 × 10 or 1 × 10 vector genomes per mouse. (A) Liver and serum samples were collected three weeks post-injection to measure the miRNA-30 expression through qPCR, with normalization to U6 RNA ( n = 6). (B) Liver tissues harvested on days 49, 63, and 77 post-injection were analyzed for hydroxyproline content ( n = 6). (C–E) qPCR to quantify the mRNA levels of α-SMA , Col1(α1) , and Col3(α1) ( n = 6). (F–H) Western blot to quantify the protein levels of α-SMA , Col1(α1) , and Col3(α1) ( n = 6). Both mRNA and protein levels of α-SMA , Col1(α1) , and Col3(α1) were normalized against GAPDH . GAPDH , 37 kDa; α-SMA , 43 kDa; Col1(α1) , 130 kDa; Col3(α1) , 117 kDa. rAAV8, recombinant adeno-associated virus serotype 8; SCR, scrambled. Statistical significance between two or more groups was evaluated using Student’s t -test and one-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Article Snippet: The membrane was then incubated with rabbit anti-GAPDH (5174S, CST, USA), anti-Col1(α1) (bs-10423R, Bioss Antibodies, USA), anti-Col3(α1) (bs-0548R, Bioss Antibodies), or anti-α-SMA (19245S, CST, USA) antibodies overnight at 4°C, followed by three 1×TBST washes.

Techniques: Over Expression, Injection, Plasmid Preparation, Expressing, Western Blot, Recombinant, Virus

Journal: Frontiers in Immunology

Article Title: Induction of hepatic fibrosis in mice with schistosomiasis by extracellular microRNA-30 derived from Schistosoma japonicum eggs

doi: 10.3389/fimmu.2024.1425384

Figure Lengend Snippet: Inhibition of miRNA-30 from S. japonicum egg-derived exosomes delays the progression of hepatic fibrosis in schistosomiasis in mice. Mice received an injection of rAAV8-SCR or rAAV8-anti-miR-30-sponge vectors at a dose of 1 × 10 virus genomes or PBS via the tail vein. After 3 weeks, these mice were dermally exposed to 20 ± 1 S. japonicum cercariae. Liver samples were collected at days 42 and 56 post-infection. (A) Granulomas and collagen deposition were assessed through Masson’s trichrome staining. (B) Expression of α-SMA at the protein level was examined on day 42 post-infection through Western blot analysis. (C) Egg count in mouse liver tissue showing there was no statistical difference between the two groups ( n = 6). (D, E) Immunohistochemical analysis on days 42 and 56 post- S. japonicum infection revealed positively stained areas for Col1(α1) , Col3(α1) , and α-SMA . (F) qPCR to quantify the mRNA levels of α-SMA , Col1(α1) , and Col3(α1) ( n = 6). All images were magnified 10-fold. Quantification of α-SMA protein levels was normalized to GAPDH . GAPDH , 37 kDa; α-SMA , 43 kDa. rAAV8, recombinant adeno-associated virus serotype 8; SCR, scrambled. Statistical significance between two or more groups was evaluated using Student’s t -test and one-way ANOVA (* P < 0.05; ** P < 0.01; *** P < 0.001. ns, no significance.

Article Snippet: The membrane was then incubated with rabbit anti-GAPDH (5174S, CST, USA), anti-Col1(α1) (bs-10423R, Bioss Antibodies, USA), anti-Col3(α1) (bs-0548R, Bioss Antibodies), or anti-α-SMA (19245S, CST, USA) antibodies overnight at 4°C, followed by three 1×TBST washes.

Techniques: Inhibition, Derivative Assay, Injection, Virus, Infection, Staining, Expressing, Western Blot, Immunohistochemical staining, Recombinant

Journal: Cancer immunology research

Article Title: MERTK inhibition cooperates with immunomodulatory cyclophosphamide to induce CXCL9 + monocyte-macrophage programming and durable antitumor immunity in triple negative breast cancer

doi: 10.1158/2326-6066.CIR-26-0278

Figure Lengend Snippet: (A) Split Umap subclustering of PMNC from 2153L CTX treated, 2153L untreated (VC), T12 CTX treated, and T12 untreated (Left to Right) scRNAseq datasets. (B) Ucell signature scoring for Phagocytosis, MHCI, MHCII, T Cell Activation, and IFN response across MP populations. (C) Schematic showing myeloid cell expressing TAMRTKs and MRX-2843 inhibition. (D) Western blot of total MerTK (tMertk), total Axl (tAxl), phospho-Erk (pErk), and β-actin from T12 and 2153L in vitro cultured cancer cells or TAM protein lysates. TAMs were isolated from tumors and cultured in vitro. (E) Non-linear regression plot of CellTiter-Glo analysis of viability in both T12 and 2153L across increasing doses of MRX-2843 to determine IC50s for each cell line. (F) Treatment strategy for CTX and MRX-2843 long-term response study. (G) 2153L tumor volume comparisons across treatments during 30 days of treatment. KM plot of overall survival across treatment groups VC (n=7), MRX-2843 (n=8), CTX (n=6), Combo (n=7). (p=0.0014, 0.0012, 0.0009, respectively) (H) T12 tumor volume comparisons across treatments during 30 days of treatment. KM plot of overall survival across treatment groups. Kaplan-Meier curves analyzed using log-rank test. VC (n=7), MRX-2843 (n=7), CTX (n=6), Combo (n=6). (p< 0.0001 for all groups). Created in BioRender. Zhao, N. (2026) https://BioRender.com/e6uqj0s .

Article Snippet: Blots were blocked with 5% non-fat dry milk (BioRad, 1706404) in PBS, then probed with the following antibodies in 5% BSA (Sigma-Aldrich, A7906) solution: MERTK (Cell Signaling Technology (CST), 38102), AXL (CST, 8661, RRID:AB_11217435), p44/42 (CST, 9102, RRID:AB_330744), p-p44/42 (CST, 9101, RRID:AB_331646), iNOS (CST, 13120, RRID:AB_2799268), PD-L1 (R&D Systems, AF1019, RRID:AB_354540), p-STAT1 (CST, 8062, RRID:AB_10949108), Arg1(CST, 93668, RRID:AB_2800207), and SOCS1 (CST, 55313).

Techniques: Activation Assay, Expressing, Inhibition, Western Blot, In Vitro, Cell Culture, Isolation, Analysis

Journal: Theranostics

Article Title: PCSK9 attenuates efferocytosis in endothelial cells and promotes vascular aging

doi: 10.7150/thno.83914

Figure Lengend Snippet: Evidence for EC efferocytosis and PCSK9 regulation of MerTK expression. ( A ) Uptake of normal or apoptotic Jurkat cells by human aortic endothelial cells (HAECs, P3) after 1 hour of co-incubation. ( B ) Efferocytosis of apoptotic Jurkat cells by P3 HAECs after 1 hour of co-incubation. ( C ) Confocal microscopy at 63x magnification verifies efferocytosis by P3 HAECs. ( D ) Efferocytosis in P3 and P15 HAECs. ( A-D ) Jurkat cells were labeled with green PKH67-GL (2 μM; Sigma-Aldrich) and HAECs were labeled with red PKH67-GL (2 μM; Sigma-Aldrich) per the manufacturer's instructions. Green cells are apoptotic Jurkat cells that were not engulfed by HAECs. Green/red small round cells are apoptotic Jurkat cells that were engulfed by HAECs. Large red cells are HAECs. ( E-F ) RNA-seq or western blot for MerTK expression in HAECs. ( G ) Expression of PCSK9 and MerTK in young (P3) and aged (P15) HAECs. ( H ) Treatment with PCSK9 or control PBS for 24 hours at indicated concentrations and MerTK expression in young P3 HAECs. ( I ) MerTK expression in young and aged HAECs treated with PBS (control) or PCSK9 at 1 µg/mL for 24 hours. ( J ) MerTK expression in P3 and P15 primary mouse aortic endothelial cells (MAECs) isolated from WT and PCSK9 -/- mice. Statistical analyses were performed with GraphPad Prism 9.0 using a two-tailed unpaired t-test. Data represent mean ± SD (n=4-6). *P< 0.05, **P<0.01, ****P< 0.0001.

Article Snippet: After blocking with 5% BSA buffer for 1 h, the membranes were incubated with primary antibody recognizing either PCSK9 (Abcam, ab31762), MerTK (Santa Cruz, sc-365499), eNOS (Cell Signaling, 32027S), NLRP3 (Cell Signaling, 15101S), IL-1β (Cell Signaling, 12703S), NF-κB (Cell Signaling, 8242S), TNF-α (Cell Signaling, 3707S), p22 phox (Cell Signaling, 37570S), or β-actin (Abcam, ab227387) at 1:1000 dilution overnight at 4°C.

Techniques: Expressing, Incubation, Confocal Microscopy, Labeling, RNA Sequencing, Western Blot, Control, Isolation, Two Tailed Test

Journal: Theranostics

Article Title: PCSK9 attenuates efferocytosis in endothelial cells and promotes vascular aging

doi: 10.7150/thno.83914

Figure Lengend Snippet: Loss of endothelial MerTK expression in aged aortic arch is restored by PCSK9 inhibition. ( A ) Immunofluorescence staining for expression of Tie2 and MerTK in the aortic arch of young (3-month-old) mice in paraffin sections. ( B ) Immunohistochemical staining for MerTK expression in aortic arch from young (3-month-old) mice, and aged (24-month-old) mice injected with saline or PCSK9 inhibitor Pep2-8. ( C-D ) Deficient expression of MerTK in aortic endothelium of aged mice is associated with increased efferocytosis in the underyling medial layer. The mice were subcutaneously administered Pep2-8 (10 µg/kg body weight in 100 µL saline) or 100 µL saline every 2 weeks for 10 weeks before reaching the age of 24 months (n=10/group).

Article Snippet: After blocking with 5% BSA buffer for 1 h, the membranes were incubated with primary antibody recognizing either PCSK9 (Abcam, ab31762), MerTK (Santa Cruz, sc-365499), eNOS (Cell Signaling, 32027S), NLRP3 (Cell Signaling, 15101S), IL-1β (Cell Signaling, 12703S), NF-κB (Cell Signaling, 8242S), TNF-α (Cell Signaling, 3707S), p22 phox (Cell Signaling, 37570S), or β-actin (Abcam, ab227387) at 1:1000 dilution overnight at 4°C.

Techniques: Expressing, Inhibition, Immunofluorescence, Staining, Immunohistochemical staining, Injection, Saline

Journal: Theranostics

Article Title: PCSK9 attenuates efferocytosis in endothelial cells and promotes vascular aging

doi: 10.7150/thno.83914

Figure Lengend Snippet: Pep2-8 treatment and disease and biology functions. Big data analytics based on gene FPKM for PCSK9 and MerTK in ( A-B ) human disease B38 GC33 and ( C-D ) mouse disease B38. ( E ) Heatmap for representative protein changes between Pep2-8 vs saline in the aortic arch of WT 24-month-old mice and MOB 1-8. ( F ) Heatmap for the disease and biology functions between Pep2-8 vs saline and MOB 1-8.

Article Snippet: After blocking with 5% BSA buffer for 1 h, the membranes were incubated with primary antibody recognizing either PCSK9 (Abcam, ab31762), MerTK (Santa Cruz, sc-365499), eNOS (Cell Signaling, 32027S), NLRP3 (Cell Signaling, 15101S), IL-1β (Cell Signaling, 12703S), NF-κB (Cell Signaling, 8242S), TNF-α (Cell Signaling, 3707S), p22 phox (Cell Signaling, 37570S), or β-actin (Abcam, ab227387) at 1:1000 dilution overnight at 4°C.

Techniques: Saline

Journal: Nature Communications

Article Title: Bacterial outer membrane vesicle based versatile nanosystem boosts the efferocytosis blockade triggered tumor-specific immunity

doi: 10.1038/s41467-023-37369-0

Figure Lengend Snippet: a Western blot analysis of phosphorylation of MerTK (p-MerTK) in M2 macrophages after mU@OMVs treatment for 2 h. b Schematic of mU@OMVs-mediated efferocytosis inhibition and antigen release. c CLMS imaging of the engulfment of AC (carboxyfluorescein succinimidyl ester (CFSE)-labeled, green) by macrophages (APC-conjugated anti-CD11b labeled, red) after a 6-h incubation. M: M2 macrophages; LC: living B16F10 cells; AC: apoptotic B16F10 cells. Scale bar: 50 μm. a , c Data are representative of three biological replicates. d , e Flow cytometry and quantification of phagocytosis after different treatments. f HMGB1 levels in the supernatants were determined by ELISA after further 18 h culture. g , h Relative abundance of tumor mutational proteins, DAMPs, predicted neoantigens and neoantigens captured by mU@OMVs. Proteins-adsorbed mU@OMVs were collected for proteomics analysis and identified according to the previous report , , . Data in e , f are presented as mean ± s.d. ( n = 3 biologically independent samples). Statistically significant differences between groups were identified by unpaired two-tailed Student’s t -test. ****P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, n.s., not significant. Source data are provided as a Source Data file.

Article Snippet: Ovalbumin (OVA), PE-conjugated OVA, Cy5-conjugated OVA, FITC-conjugated anti- E. coli LPS antibody (catalog no. bs-8000R-FITC), anti-phospho-MerTK antibody (catalog no. bs-18791R), anti-phospho-Axl antibody (catalog no. bs-5181R), and anti-Axl antibody (catalog no. bs-5180R) were purchased from Bioss (China).

Techniques: Western Blot, Inhibition, Imaging, Labeling, Incubation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test