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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies.

doi: 10.4049/jimmunol.2200597

Figure Lengend Snippet: FIGURE 1. Phagocytosis and efferocytosis activities of mouse macrophage cell line J774A.1. (A) Kinetic curve of phagocytosis of pHrodo-labeled E. coli bioparticles (EcBp) by J774A.1 cells (gray squares) with representative images (original magnification ×10) taken at 0 h and 17 h (see inlets) and background fluorescence emitted by pHrodo-labeled E. coli bioparticles alone (white squares). (B) Representative images in brightfield and fluorescent channels (original magnification ×10) captured by Incucyte at different time points after adding pHrodo redlabeled apoptotic Jurkat cells to J774A.1 cells. (C) Kinetic curves showing the red fluorescence captured in J774A.1 cells after feeding pHrodo redlabeled apoptotic Jurkat cells (red circles) and pHrodo-labeled healthy Jurkat cells (blue diamonds) as well as background fluorescence emitted by apoptotic (white circles) and healthy (white diamonds) Jurkat cells alone. (D) Live-cell images (original magnification ×20) obtained by the ImageXpress confocal high-content imaging system after feeding LysoTracker (green)- treated J774A.1 cells with pHrodo redlabeled apoptotic Jurkat cells. Images in red fluorescence (top panel), green fluorescence (middle panel), and merged with brightfield (bottom panel) showing the colocalization of engulfed pHrodo-labeled apop-totic Jurkat cells with the lysosomal compartment of J774A.1 cells. Only the macrophage in the right has the engulfed apoptotic meal. (E) Flow cytometry analyses of efferocytosis receptors Tyro3 (green), Axl (blue), and MerTK (red) in J774A.1 cells, overlaid with the background staining of the isotype control (gray). Kinetic curves show representative data (mean ± SD; n 5 4) of three independent experiments.

Article Snippet: The following reagents were used in flow cytometry: PE anti-mouse CD16.2 (BioLegend, 149504), PE anti-mouse CD64 (BioLegend, 139304), PE anti-mouse MerTK (BioLegend, 151506), FITC anti-mouse F4/80 (BioLegend, 123108), FITC anti-mouse CD163, PE anti-mouse CD206 (BioLegend, 141707), allophycocyanin anti-mouse CD80 (BioLegend, 104713), allophycocyanin anti-mouse CD68 (BioLegend, 137007), PE anti-mouse CD11c (BioLegend, 117307), PE anti-human MerTK (R&D Systems, FAB8912P), PE anti-human CD68 (BioLegend, 333807), allophycocyanin anti-human CD16 (BioLegend, 302011), PE anti-human CD32 (BioLegend, 303205), allophycocyanin anti-human CD64 (BioLegend, 305013), allophycocyanin anti-human CD163 (BioLegend, 333610), allophycocyanin anti-human CD206 (BioLegend, 321110), allophycocyanin anti-human CD80 (BioLegend, 305219), PE anti-human CD86 (BioLegend, 305405), FITC annexin V (BioLegend, 640905), allophycocyanin anti-human/anti-mouse TREM2 (R&D Systems, FAB17291A).

Techniques: Labeling, Fluorescence, Imaging, Flow Cytometry, Staining, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies.

doi: 10.4049/jimmunol.2200597

Figure Lengend Snippet: FIGURE 2. Screening anti-murine MerTK Abs using mouse peritoneal macrophages. (A) Flow cytometric analyses of mouse peritoneal macrophages. (B) Kinetic curve of efferocytosis of pHrodo redlabeled apoptotic Jurkat cells by mouse peritoneal macrophages. (C) Single-point pAKT homogeneous time-resolved fluorescence (HTRF) screening of anti-murine MerTK Abs (gray), with positive control Ab AF591 (green) and isotype control (white) using mouse peritoneal macrophages, treated with Gas6-Fc. (D) Two anti-murine MerTK Abs (purple and red) showed dose-dependent inhibitory activities in pAKT HTRF assay, with an anti-MerTK control Ab AF591 (green) and an isotype control (gray). (E) Two anti-murine MerTK Abs (purple and red) showed dose-dependent inhibitory activities of efferocytosis mediated by mouse peritoneal macrophages. pAKT HTRF (D) and efferocytosis (E) curves show representative data (mean ± SD; n 5 3) of three independent experiments, respectively.

Article Snippet: The following reagents were used in flow cytometry: PE anti-mouse CD16.2 (BioLegend, 149504), PE anti-mouse CD64 (BioLegend, 139304), PE anti-mouse MerTK (BioLegend, 151506), FITC anti-mouse F4/80 (BioLegend, 123108), FITC anti-mouse CD163, PE anti-mouse CD206 (BioLegend, 141707), allophycocyanin anti-mouse CD80 (BioLegend, 104713), allophycocyanin anti-mouse CD68 (BioLegend, 137007), PE anti-mouse CD11c (BioLegend, 117307), PE anti-human MerTK (R&D Systems, FAB8912P), PE anti-human CD68 (BioLegend, 333807), allophycocyanin anti-human CD16 (BioLegend, 302011), PE anti-human CD32 (BioLegend, 303205), allophycocyanin anti-human CD64 (BioLegend, 305013), allophycocyanin anti-human CD163 (BioLegend, 333610), allophycocyanin anti-human CD206 (BioLegend, 321110), allophycocyanin anti-human CD80 (BioLegend, 305219), PE anti-human CD86 (BioLegend, 305405), FITC annexin V (BioLegend, 640905), allophycocyanin anti-human/anti-mouse TREM2 (R&D Systems, FAB17291A).

Techniques: Fluorescence, Positive Control, Control, HTRF Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies.

doi: 10.4049/jimmunol.2200597

Figure Lengend Snippet: FIGURE 3. Characterization of in vitro differentiated human macrophages. (A) Expression of Tyro3 (green), Axl (blue), and MerTK (red) on human cell line U937 and in vitro differentiated human M1 and M2 macrophages by flow cytometry. (B) Different expression of cell surface markers CD163 and MerTK and distinct phenotype between in vitro differentiated human M1 and M2 macrophages (brightfield, original magnification ×10). (C, D) Comparative gene expression profiles (C) and cytokine profiles (D) of in vitro differentiated human M1 (red) and M2 (blue) macrophages after LPS treatment. (E) Efferocytosis kinetic curves of pHrodo-labeled apoptotic Jurkat cells by human M1 (red diamonds) and human M2 macrophages (blue circles) with assay background (white diamonds, pHrodo-labeled apoptotic Jurkat cells only). Gene expression (C) and cytokine profiles (D) show representative data (mean ± SD; n 5 3) of two independent experiments. Efferocytosis kinetic curves (E) show representative data (mean ± SD; n 5 4) of two independent experiments.

Article Snippet: The following reagents were used in flow cytometry: PE anti-mouse CD16.2 (BioLegend, 149504), PE anti-mouse CD64 (BioLegend, 139304), PE anti-mouse MerTK (BioLegend, 151506), FITC anti-mouse F4/80 (BioLegend, 123108), FITC anti-mouse CD163, PE anti-mouse CD206 (BioLegend, 141707), allophycocyanin anti-mouse CD80 (BioLegend, 104713), allophycocyanin anti-mouse CD68 (BioLegend, 137007), PE anti-mouse CD11c (BioLegend, 117307), PE anti-human MerTK (R&D Systems, FAB8912P), PE anti-human CD68 (BioLegend, 333807), allophycocyanin anti-human CD16 (BioLegend, 302011), PE anti-human CD32 (BioLegend, 303205), allophycocyanin anti-human CD64 (BioLegend, 305013), allophycocyanin anti-human CD163 (BioLegend, 333610), allophycocyanin anti-human CD206 (BioLegend, 321110), allophycocyanin anti-human CD80 (BioLegend, 305219), PE anti-human CD86 (BioLegend, 305405), FITC annexin V (BioLegend, 640905), allophycocyanin anti-human/anti-mouse TREM2 (R&D Systems, FAB17291A).

Techniques: In Vitro, Expressing, Flow Cytometry, Gene Expression, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies.

doi: 10.4049/jimmunol.2200597

Figure Lengend Snippet: FIGURE 4. Optimization of efferocytosis assay using in vitro differentiated human M2 macrophages. (A) Efferocytosis kinetic curves at different macrophage/ apoptotic cell ratios. (B) Comparison of the efferocytosis inhibition curves of anti-MerTK AF891 using regular (gray) versus slow-speed centrifugation (white) to remove cell debris from apoptotic meal; forward/side scatter flow analysis (inlets) showed an 80% decrease of cell debris in apoptotic meal after cleanup. (C) Comparison of the efferocytosis inhibition curves of anti-MerTK AF891 using the default (white) versus optimized (green) image analysis algorithm (IAA). Inlets (original magnification ×10) show the detection of autofluorescence from pHrodo redlabeled cells alone with the default IAA (blue dots, top inlet) and optimized IAA (bottom inlet). (D) Dose-dependent inhibitory activities of anti-MerTK Ab (green) and cytochalasin D (purple) in human M2 macrophagemediated efferocytosis. (E) Variability of signal across a 96-well plate (top panel) is significantly improved after normalization by macrophage numbers/image (bottom panel); inlets show representative brightfield images (original magnification ×10) of cell seeding variability with cell detection algorithm (yellow outlines). Anti-MerTK AF891 was used to block efferocytosis (gray), and normal goat IgG (from R&D Systems) was used as a negative control (red and green). (F) Comparison of anti-MerTK efferocytosis inhibitory activities of frozen human M2 (blue) versus freshly differentiated M2 macrophages (green). Efferocytosis activity curves show representative data (mean ± SD; n 5 4) of at least two independent experiments.

Article Snippet: The following reagents were used in flow cytometry: PE anti-mouse CD16.2 (BioLegend, 149504), PE anti-mouse CD64 (BioLegend, 139304), PE anti-mouse MerTK (BioLegend, 151506), FITC anti-mouse F4/80 (BioLegend, 123108), FITC anti-mouse CD163, PE anti-mouse CD206 (BioLegend, 141707), allophycocyanin anti-mouse CD80 (BioLegend, 104713), allophycocyanin anti-mouse CD68 (BioLegend, 137007), PE anti-mouse CD11c (BioLegend, 117307), PE anti-human MerTK (R&D Systems, FAB8912P), PE anti-human CD68 (BioLegend, 333807), allophycocyanin anti-human CD16 (BioLegend, 302011), PE anti-human CD32 (BioLegend, 303205), allophycocyanin anti-human CD64 (BioLegend, 305013), allophycocyanin anti-human CD163 (BioLegend, 333610), allophycocyanin anti-human CD206 (BioLegend, 321110), allophycocyanin anti-human CD80 (BioLegend, 305219), PE anti-human CD86 (BioLegend, 305405), FITC annexin V (BioLegend, 640905), allophycocyanin anti-human/anti-mouse TREM2 (R&D Systems, FAB17291A).

Techniques: In Vitro, Comparison, Inhibition, Centrifugation, Blocking Assay, Negative Control, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies.

doi: 10.4049/jimmunol.2200597

Figure Lengend Snippet: FIGURE 5. Screening and characterization of anti-human MerTK Abs using established human efferocytosis assay. (A) Representative Ab screening results showed various anti-MerTK Abs with distinct potencies and maximum inhibitory activities. (B) Correlation of Ab potencies (IC50) between macro- phages differentiated from two different donors’ CD141 monocytes. (C) A subset of anti-MerTK Abs showed consistent inhibitory potency ranking in effero- cytosis assays using human M2 macrophages differentiated from three different donors. Efferocytosis activity curves show representative data (mean ± SEM; n 5 4) of three independent experiments.

Article Snippet: The following reagents were used in flow cytometry: PE anti-mouse CD16.2 (BioLegend, 149504), PE anti-mouse CD64 (BioLegend, 139304), PE anti-mouse MerTK (BioLegend, 151506), FITC anti-mouse F4/80 (BioLegend, 123108), FITC anti-mouse CD163, PE anti-mouse CD206 (BioLegend, 141707), allophycocyanin anti-mouse CD80 (BioLegend, 104713), allophycocyanin anti-mouse CD68 (BioLegend, 137007), PE anti-mouse CD11c (BioLegend, 117307), PE anti-human MerTK (R&D Systems, FAB8912P), PE anti-human CD68 (BioLegend, 333807), allophycocyanin anti-human CD16 (BioLegend, 302011), PE anti-human CD32 (BioLegend, 303205), allophycocyanin anti-human CD64 (BioLegend, 305013), allophycocyanin anti-human CD163 (BioLegend, 333610), allophycocyanin anti-human CD206 (BioLegend, 321110), allophycocyanin anti-human CD80 (BioLegend, 305219), PE anti-human CD86 (BioLegend, 305405), FITC annexin V (BioLegend, 640905), allophycocyanin anti-human/anti-mouse TREM2 (R&D Systems, FAB17291A).

Techniques: Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies.

doi: 10.4049/jimmunol.2200597

Figure Lengend Snippet: FIGURE 6. Efferocytosis assay using cynomolgus monkey M2 macrophages. (A) In vitro differentiated cynomolgus monkey M2 macrophages (brightfield, original magnification ×10) showed phenotypes similar to those of their human counterparts. (B) A representative kinetic curve (purple) of efferocytosis mediated by monkey M2 macrophages with an image (inlet, original magnification ×10) taken at 4.5 h; assay background (pHrodo-labeled apoptotic Jurkat cells only) is shown in white circles. (C) One anti-MerTK Ab showed comparable inhibitory potencies in monkey (purple circles) versus human (blue circles) M2-mediated efferocytosis; isotype control Ab shown in gray (circles and squares represent results in monkey and human M2-mediated effero- cytosis, respectively). Efferocytosis activity curves showed representative data (mean ± SD; n 5 4) of three independent experiments.

Article Snippet: The following reagents were used in flow cytometry: PE anti-mouse CD16.2 (BioLegend, 149504), PE anti-mouse CD64 (BioLegend, 139304), PE anti-mouse MerTK (BioLegend, 151506), FITC anti-mouse F4/80 (BioLegend, 123108), FITC anti-mouse CD163, PE anti-mouse CD206 (BioLegend, 141707), allophycocyanin anti-mouse CD80 (BioLegend, 104713), allophycocyanin anti-mouse CD68 (BioLegend, 137007), PE anti-mouse CD11c (BioLegend, 117307), PE anti-human MerTK (R&D Systems, FAB8912P), PE anti-human CD68 (BioLegend, 333807), allophycocyanin anti-human CD16 (BioLegend, 302011), PE anti-human CD32 (BioLegend, 303205), allophycocyanin anti-human CD64 (BioLegend, 305013), allophycocyanin anti-human CD163 (BioLegend, 333610), allophycocyanin anti-human CD206 (BioLegend, 321110), allophycocyanin anti-human CD80 (BioLegend, 305219), PE anti-human CD86 (BioLegend, 305405), FITC annexin V (BioLegend, 640905), allophycocyanin anti-human/anti-mouse TREM2 (R&D Systems, FAB17291A).

Techniques: In Vitro, Labeling, Control, Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies.

doi: 10.4049/jimmunol.2200597

Figure Lengend Snippet: FIGURE 4. Optimization of efferocytosis assay using in vitro differentiated human M2 macrophages. (A) Efferocytosis kinetic curves at different macrophage/ apoptotic cell ratios. (B) Comparison of the efferocytosis inhibition curves of anti-MerTK AF891 using regular (gray) versus slow-speed centrifugation (white) to remove cell debris from apoptotic meal; forward/side scatter flow analysis (inlets) showed an 80% decrease of cell debris in apoptotic meal after cleanup. (C) Comparison of the efferocytosis inhibition curves of anti-MerTK AF891 using the default (white) versus optimized (green) image analysis algorithm (IAA). Inlets (original magnification ×10) show the detection of autofluorescence from pHrodo redlabeled cells alone with the default IAA (blue dots, top inlet) and optimized IAA (bottom inlet). (D) Dose-dependent inhibitory activities of anti-MerTK Ab (green) and cytochalasin D (purple) in human M2 macrophagemediated efferocytosis. (E) Variability of signal across a 96-well plate (top panel) is significantly improved after normalization by macrophage numbers/image (bottom panel); inlets show representative brightfield images (original magnification ×10) of cell seeding variability with cell detection algorithm (yellow outlines). Anti-MerTK AF891 was used to block efferocytosis (gray), and normal goat IgG (from R&D Systems) was used as a negative control (red and green). (F) Comparison of anti-MerTK efferocytosis inhibitory activities of frozen human M2 (blue) versus freshly differentiated M2 macrophages (green). Efferocytosis activity curves show representative data (mean ± SD; n 5 4) of at least two independent experiments.

Article Snippet: Anti-MerTK AF891 was used to block efferocytosis (gray), and normal goat IgG (from R&D Systems) was used as a negative control (red and green). (F) Comparison of anti-MerTK efferocytosis inhibitory activities of frozen human M2 (blue) versus freshly differentiated M2 macrophages (green).

Techniques: In Vitro, Comparison, Inhibition, Centrifugation, Blocking Assay, Negative Control, Activity 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: 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: JCI insight

Article Title: Sitravatinib potentiates immune checkpoint blockade in refractory cancer models.

doi: 10.1172/jci.insight.124184

Figure Lengend Snippet: Figure 1. MerTK inhibition with sitravatinib directly affects macrophage phenotype. The expression of M1-type macrophage markers Tnfα, Il-6, and Il-12 (A) and M2-type macro- phage markers Arg1, Ym-1, and Fizz-1 (B) in bone marrow–derived macrophages (BMDMs). BMDMs were harvested from WT C57BL/6 or MerTK–/– (green) mice, stimulated with 20 ng/ ml LPS for 2 hours (A) or 40 ng/ml IL-4 for 18 hours (B). Each stimulation was performed with or without sitravatinib (12.5, 50, 200, and 800 nM) in the presence (red and green) or absence (blue) of KLN205 conditioned media (CM). The expression level of TNF-α, IL-6, IL-12, arginase 1, YM-1, and Fizz-1 was determined by q-PCR. Three independent experiments using duplicate samples were performed. Data are displayed as fold change normalized to control in each condition (mean ± SD). For each marker, the top graph is the basal expression change in each stimulation condition, and the bottom graph is expression change caused by different con- centrations of sitravatinib in each condition. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 vs. the control (WT macrophages without stimulation) or DMSO (0 nM) in each condition by ANOVA.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibody, p-MerTK (Novus Biologicals, NB300-690), MerTK (R&D Systems, AF591), Actin (MilliporeSigma, A2066), Axl (Santa Cruz, sc-1096), and PDGFRα (Cell Signaling, 3174), followed by corresponding horseradish peroxidase–conjugated secondary antibody (Jackson ImmunoResearch).

Techniques: Inhibition, Expressing, Derivative Assay, Control, Marker

Journal: JCI insight

Article Title: Sitravatinib potentiates immune checkpoint blockade in refractory cancer models.

doi: 10.1172/jci.insight.124184

Figure Lengend Snippet: Figure 3. Sitravatinib potently inhibits MerTK activity and reduces angiogenesis. (A) Lysates of tumors from KLN205 tumor-bearing animals treated with control (Ctrl), sitravatinib, or glesatinib were probed for the indicated targets by Western blotting. (B) KLN205 tumor– bearing animals treated with control, sitravatinib (sitra), or glesatinib (gles) were evaluated by immunohistochem- istry for the expression level of the indicated markers. Images were taken by Nanozoomer and analyzed using ImageJ. Quantification of percentage of the area analyzed positive for staining (% area fraction) is shown. Data are displayed as mean ± SD and represent images covering the whole tumor, with 4–5 animals per group analyzed. Original magnification, ×20. **P < 0.01, ****P < 0.001 vs. control by ANOVA.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibody, p-MerTK (Novus Biologicals, NB300-690), MerTK (R&D Systems, AF591), Actin (MilliporeSigma, A2066), Axl (Santa Cruz, sc-1096), and PDGFRα (Cell Signaling, 3174), followed by corresponding horseradish peroxidase–conjugated secondary antibody (Jackson ImmunoResearch).

Techniques: Activity Assay, Control, Western Blot, Expressing, Staining

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: The MerTK flox/flox mice and Tie2-Cre mice on the C57BL/6J background were generated by Cyagen US (Santa Clara, CA) and housed in the Division of Laboratory Animal Medicine at our institution with an approved ethic number of A23050.

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: The MerTK flox/flox mice and Tie2-Cre mice on the C57BL/6J background were generated by Cyagen US (Santa Clara, CA) and housed in the Division of Laboratory Animal Medicine at our institution with an approved ethic number of A23050.

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: The MerTK flox/flox mice and Tie2-Cre mice on the C57BL/6J background were generated by Cyagen US (Santa Clara, CA) and housed in the Division of Laboratory Animal Medicine at our institution with an approved ethic number of A23050.

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: The MerTK flox/flox mice and Tie2-Cre mice on the C57BL/6J background were generated by Cyagen US (Santa Clara, CA) and housed in the Division of Laboratory Animal Medicine at our institution with an approved ethic number of A23050.

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: The MerTK flox/flox mice and Tie2-Cre mice on the C57BL/6J background were generated by Cyagen US (Santa Clara, CA) and housed in the Division of Laboratory Animal Medicine at our institution with an approved ethic number of A23050.

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: The MerTK flox/flox mice and Tie2-Cre mice on the C57BL/6J background were generated by Cyagen US (Santa Clara, CA) and housed in the Division of Laboratory Animal Medicine at our institution with an approved ethic number of A23050.

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: The MerTK flox/flox mice and Tie2-Cre mice on the C57BL/6J background were generated by Cyagen US (Santa Clara, CA) and housed in the Division of Laboratory Animal Medicine at our institution with an approved ethic number of A23050.

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: The MerTK flox/flox mice and Tie2-Cre mice on the C57BL/6J background were generated by Cyagen US (Santa Clara, CA) and housed in the Division of Laboratory Animal Medicine at our institution with an approved ethic number of A23050.

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: The MerTK flox/flox mice and Tie2-Cre mice on the C57BL/6J background were generated by Cyagen US (Santa Clara, CA) and housed in the Division of Laboratory Animal Medicine at our institution with an approved ethic number of A23050.

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

Journal: The Journal of Clinical Investigation

Article Title: MERTK receptor tyrosine kinase is a therapeutic target in melanoma

doi: 10.1172/JCI67816

Figure Lengend Snippet: (A) TAM receptor expression in 4 metastatic melanoma cell lines. Lysates from HMCB, SKMEL119, G361, and SKMEL5 were analyzed by Western blot for MERTK, TYRO3, and AXL expression. GAPDH was detected as a loading control. (B) MERTK phosphorylation in melanoma cell lines. Subconfluent cultures were treated with pervanadate. MERTK was immunoprecipitated (IP) from whole-cell lysates then visualized by Western blot for phosphorylated MERTK. As a control, total MERTK protein was detected in whole-cell lysates corresponding to the protein load applied to the immunoprecipitation. (C) GAS6 activation of signaling pathways in melanoma cell lines. Phosphokinase array suggests p38, ERK1/2, GSK3α/β, AKT, AMPK, STAT5, CHK-2, FAK, and STAT6 as signaling molecules downstream of MERTK. Relative phosphorylation between control-treated and GAS6-treated cells is shown graphically for a subset of signaling molecules probed. (D) Western blot of phospho- and total STAT6, AKT, and ERK1/2. Cell cultures were stimulated with GAS6 and whole-cell lysates were evaluated by Western blot analysis. Actin was detected as a loading control.

Article Snippet: For phospho-MERTK assays, subconfluent cultures were treated with pervanadate solution for 3 minutes, MERTK was immunoprecipitated from whole-cell lysates (MAB8912; R&D Systems) then visualized with Western blot using p-MERTK antibody (PhosphoSolutions) ( 11 ).

Techniques: Expressing, Western Blot, Control, Phospho-proteomics, Immunoprecipitation, Activation Assay, Protein-Protein interactions

Journal: The Journal of Clinical Investigation

Article Title: MERTK receptor tyrosine kinase is a therapeutic target in melanoma

doi: 10.1172/JCI67816

Figure Lengend Snippet: (A) Chemical structure of UNC1062. (B) Inhibition of phospho-MERTK with UNC1062. Subconfluent cultures were treated with indicated doses of UNC1062 for 90 minutes followed by pervanadate treatment. MERTK was immunoprecipitated from whole-cell lysates then visualized by Western blot for phosphorylated MERTK and total MERTK. (C) Downstream signaling inhibition after administration of UNC1062. Cultures were serum starved then treated with UNC1062 for 90 minutes. After treatment with UNC1062, cells were stimulated with GAS6 and lysates were analyzed by Western blot for phospho- and total STAT6, AKT, and ERK1/2. GAPDH was detected as a loading control.

Article Snippet: For phospho-MERTK assays, subconfluent cultures were treated with pervanadate solution for 3 minutes, MERTK was immunoprecipitated from whole-cell lysates (MAB8912; R&D Systems) then visualized with Western blot using p-MERTK antibody (PhosphoSolutions) ( 11 ).

Techniques: Inhibition, Immunoprecipitation, Western Blot, Control

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