Journal: Frontiers in Immunology

Article Title: YiQi-HuoXue prescription ameliorates LPS-induced sepsis-associated encephalopathy via VCAM-1–mediated microglial efferocytosis

doi: 10.3389/fimmu.2026.1792688

Figure Lengend Snippet: Pharmacological modulation of MERTK and VCAM1 implicates a VCAM1-linked efferocytosis and anti-inflammatory mechanism for YQHXP. (A) Flow cytometry analysis of PKH67 + /F4/80 + double-positive cells in BV2–HT22 co-culture across seven groups. (B) Quantification of efferocytosis rate(n=3). (C) Flow cytometry plots of apoptotic BV2 cells (Annexin V/PI staining). (D) Quantification of apoptosis rate(n=3). (E) Western blot and quantification of C1QB protein expression(n=3). (F) Western blot and quantification of MERTK protein expression(n=3). (G) Western blot and quantification of VCAM1 protein expression(n=3). (H) TNF-α levels in BV2 supernatant measured by ELISA(n=3). IL-6 levels in BV2 supernatant measured by ELISA(n=3). *P < 0.05 relative to the model group; **P < 0.01, ***P < 0.001 relative to the model group. # P < 0.05 relative to the UNC2250 group; ## P < 0.01 relative to the UNC2250 group; ### P < 0.001 relative to the UNC2250 group.

Article Snippet: The MERTK inhibitor UNC2250 (Cat. HY-15797), rutin (Cat. HY-N0148), and Ginsenoside Rg1 (Cat. HY-N0045) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Flow Cytometry, Co-Culture Assay, Staining, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Translational Neurodegeneration

Article Title: Attenuating α-synuclein pathology in mice with in situ engineered astrocytes

doi: 10.1186/s40035-025-00518-0

Figure Lengend Snippet: Expression of CAR-A and effect on α-synO phagocytosis and digestion. a Design of the CAR-expression plasmid. CAR was expressed in fusion with 3A scFv and enhanced green fluorescent protein (EGFP) under the control of the astrocyte-specific promotor GfaABC1D. SP, signal peptide; Poly(A), polyadenylation signal; ORI, origin of replication; KanR, kanamycin resistance gene. b Representative image of CAR expression on an astrocyte. The co-localization of 3A, MerTK and EGFP was assessed by confocal microscopy. Scale bars, 10 μm. c Flow cytometry analysis of the binding of CAR-A and ns-CAR-A to α-syn monomers and oligomers (α-synOs). The astrocytes were transfected with CAR or ns-CAR lipoplexes for 48 h. After 2 h-incubation with 1 μmol/L α-syn monomers (α-syn) or α-synOs, cells were stained with PE-labeled anti-α-syn antibody. d PE fluorescence in EGFP-positive astrocytes. n = 3 independent experiments. e Flow cytometry analysis of the amount of α-synO engulfed by CAR-A, ns-CAR-A and NC-A in the presence of different α-synO concentrations. n = 3 independent experiments. f Representative images depicting the phases of engulfment and digestion of α-synO by CAR-A. CAR-A was treated with 1 μmol/L α-synO, and the medium was changed after 1 h incubation. α-SynO and Lamp1 in CAR-A were stained with respective antibodies at different time points and imaged by confocal microscopy. Scale bars, 5 μm. g The kinetic curves of α-synO digestion in CAR-A, ns-CAR-A and NC-A. n = 3 independent experiments. h Statistical analysis of the proportion of α-syn colocalized with Lamp1 in digestion stage in ( f ) by Image J. n = 4 independent experiments. i Intracellular α-syn in Triton X-100-soluble and -insoluble fraction detected by Western blotting at different time points post astrocytic phagocytose of α-synOs. β-actin was used as a control. j Quantification of α-syn ( i ) using Image J. n = 3 independent experiments. k Representative images depicting the binding of ns-CAR-A, NC-A and CAR-A to α-syn monomers and oligomers. Scale bars, 5 μm. Data are mean ± S.E.M. One-way ANOVA ( d ) or Two-way ANOVA ( e ) followed by Tukey’s multiple comparison test was used for statistical analysis. * P < 0.05, ** P < 0.01, **** P < 0.0001 indicate significance compared to respective groups

Article Snippet: In the first construct, the enhanced CMV promotor sequence in the MerTK expression plasmid (purchased from Sino Biological Inc., #MG50514-ACG) was replaced with GfaABC1D promotor sequence synthesized from Sangon.

Techniques: Expressing, Plasmid Preparation, Control, Confocal Microscopy, Flow Cytometry, Binding Assay, Transfection, Incubation, Staining, Labeling, Fluorescence, Western Blot, Comparison

Journal: Journal of Translational Medicine

Article Title: Hypoxic migrasomes drive colorectal cancer liver metastasis by mediating CD5L + macrophage efferocytosis via NRP2/PROX1 axis

doi: 10.1186/s12967-025-07485-0

Figure Lengend Snippet: Phagocytic activity and immunofluorescence validation of CD5L⁺ macrophages in CRC liver metastases following migrasome treatment. ( A ) UMAP blot showing the expression of migrasome marker TSPAN4 in myeloid subsets. ( B ) Boxplot showing efferocytosis scores across the 10 identified myeloid cell subtypes. ( C ) Violin plots depicting the expression of efferocytosis markers CD300B, MERTK, and CD300D across 10 distinct myeloid cell subtypes. ( D ) UMAP plots displaying the expression patterns of three efferocytosis-associated marker genes specifically enriched in CD5L⁺ macrophages. ( E ) Immunofluorescence staining of tumor tissues from MC38-tumor bearing mice showing colocalization of CD163, CD5L, and the migrasome marker MERTK in both treatment groups. Increased MERTK expression is observed in the hypoxic group, indicating enhanced migrasome targeting of CD5L⁺ macrophages

Article Snippet: Samples were incubated with primary antibodies, CD5L (1:500, 17224-1-AP, Proteintech), CD163 (1:200, ab182422, Abcam), NRP2 (1:250, #3366, CST), PROX1(1:250, sc-81983, Santa cruz), MERTK (200 μg/mL, sc-365499, Santa Cruz).

Techniques: Activity Assay, Immunofluorescence, Biomarker Discovery, Expressing, Marker, Staining

Journal: Journal of Translational Medicine

Article Title: Hypoxic migrasomes drive colorectal cancer liver metastasis by mediating CD5L + macrophage efferocytosis via NRP2/PROX1 axis

doi: 10.1186/s12967-025-07485-0

Figure Lengend Snippet: Migrasomal NRP2 is required for CRC-induced CD5L⁺ macrophage differentiation and efferocytosis. ( A ) RT-qPCR analysis confirming efficient knockdown of NRP2 in MC38 cells under hypoxia. ( B ) Western blot analysis confirming efficient knockdown of NRP2 in MC38 cells under hypoxia. ( C ) Flow cytometry analysis of CD5L⁺ macrophage proportion after treatment with control or NRP2-deficient hypoxic migrasomes from MC38 cells. ( D ) Immunofluorescence assay of efferocytosis by CD5L⁺ macrophages following treatment with control or NRP2-deficient hypoxic migrasomes. F4/80 (green) labels macrophages; PI (red) labels apoptotic tumor cells. Scare bar: 50 μm. ( E ) Quantification of mRNA expression of efferocytosis receptors (MERTK, TYRO3, OLR1, CD36, AXL, and TIM3) in macrophages treated with control or NRP2-deficient migrasomes by RT-qPCR. ( F ) Quantification of protein expression of efferocytosis receptors (MERTK, TYRO3, OLR1, CD36, AXL, and TIM3) in macrophages treated with control or NRP2-deficient migrasomes by Western blot. * p < 0.05, ** p < 0.01

Article Snippet: Samples were incubated with primary antibodies, CD5L (1:500, 17224-1-AP, Proteintech), CD163 (1:200, ab182422, Abcam), NRP2 (1:250, #3366, CST), PROX1(1:250, sc-81983, Santa cruz), MERTK (200 μg/mL, sc-365499, Santa Cruz).

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Flow Cytometry, Control, Immunofluorescence, Expressing

Journal: Journal of Translational Medicine

Article Title: Hypoxic migrasomes drive colorectal cancer liver metastasis by mediating CD5L + macrophage efferocytosis via NRP2/PROX1 axis

doi: 10.1186/s12967-025-07485-0

Figure Lengend Snippet: NRP2–PROX1 interaction promotes CD5L⁺ macrophage differentiation and enhances efferocytosis. ( A ) Co-immunoprecipitation (Co-IP) assays showing that NRP2 interacts with PROX1 in macrophages under normoxic and hypoxic migrasome-treated conditions. ( B ) Immunofluorescence co-localization images confirming the spatial association between NRP2 (green) and PROX1 (red) in macrophages. Nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. ( C ) Flow cytometry analysis showing the proportion of CD5L⁺ macrophages following NRP2 overexpression and/or PROX1 knockdown. ( D ) RT-qPCR analysis of efferocytosis-related genes (AXL, MERTK, and TYRO3) in macrophages with indicated treatments. ( E ) Immunofluorescence staining of F4/80⁺ macrophages (green) engulfing PI-labeled apoptotic MC38 debris (red). Knockdown of PROX1 suppressed efferocytic activity and attenuated the NRP2-induced enhancement. Scale bar, 50 μm. ( F ) Representative fluorescence images and quantification of transwell assay. Fluorescently labeled CRC cells were co-cultured with macrophages overexpressing NRP2, MERTK-knockdown macrophages, or macrophages with combined NRP2 overexpression and MERTK knockdown, and CRC cell transmigration was assessed using a transwell assay. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Samples were incubated with primary antibodies, CD5L (1:500, 17224-1-AP, Proteintech), CD163 (1:200, ab182422, Abcam), NRP2 (1:250, #3366, CST), PROX1(1:250, sc-81983, Santa cruz), MERTK (200 μg/mL, sc-365499, Santa Cruz).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Immunofluorescence, Flow Cytometry, Over Expression, Knockdown, Quantitative RT-PCR, Staining, Labeling, Activity Assay, Fluorescence, Transwell Assay, Cell Culture, Transmigration 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 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: Arthritis Research & Therapy

Article Title: Interleukin 37 limits monosodium urate crystal-induced innate immune responses in human and murine models of gout

doi: 10.1186/s13075-016-1167-y

Figure Lengend Snippet: Contribution of the Mertk inhibitor to the IL-37-mediated anti-inflammatory effect in monosodium urate ( MSU )-induced models in vitro and in vivo. a – c Concentration of secreted IL-1β, IL-8 and CCL2 in THP-1 macrophages treated with or without recombinant human IL-37 (rhIL-37) for 3 h, followed by incubation for 1 h with or without Mertk inhibitor and then incubated with lipopolysaccharide (LPS) or MSU separately for a further 18 h; * P < 0.05. d Different dosage of rhIL-37 was given preventively or therapeutically with or without Mertk inhibitor intervention in mice with gouty arthritis, and foot thickness was evaluated; * P < 0.05. e , f Histopathological analysis by H&E staining in a joint from the group treated with rhIL-37 treatment and Mertk inhibitor intervention (×100 original magnification ( e ) and × 200 original magnification ( f ); arrow inflammation in soft tissue and joint space. g – k The protein level of Smad3, IL-1R8, S​OCS3 and NLRP3 was verified by western blotting in the IL-37 treatment groups with or without Mertk inhibitor intervention. Protein levels in different groups were expressed as a ratio to that of corresponding glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ); * P < 0.05 ** P < 0.01

Article Snippet: THP-1-derived macrophages were treated with or without 10 ng/ml recombinant human IL-37 (rhIL-37; R&D Systems, Minneapolis, MN, USA) for 3 h, followed by incubation for 1 h with or without a small-molecule inhibitor of Mertk (Mertk inhibitor UNC2250, 20 nM; Selleckchem, Houston, TX, USA) and then incubated with either 1 μg/ml lipopolysaccharide (LPS) (Sigma), 5 mM ATP (Sigma), or MSU (50 μg/ml, 100 μg/ml) separately for a further 18 h. Culture supernatants were harvested and frozen at −80 °C for later cytokine analysis by ELISA.

Techniques: In Vitro, In Vivo, Concentration Assay, Recombinant, Incubation, Staining, Western Blot

Journal: Cancers

Article Title: Targeting Tyro3, Axl, and MerTK Receptor Tyrosine Kinases Significantly Sensitizes Triple-Negative Breast Cancer to CDK4/6 Inhibition

doi: 10.3390/cancers16122253

Figure Lengend Snippet: TAM/Met receptor tyrosine kinases are upregulated in TNBC. ( a ) Schematic representation of receptor tyrosine kinase-mediated regulation of CDK4/6. ( b , c ) Immunoblot was performed on cell lines treated for 24 h with Abe (2 μM) ( b ) and for 25 min with either HGF (40 ng/mL) or Gas6 (400 ng/mL) ( c ). Protein levels were determined for phospho-AXL and phospho−MET. ( d ) Comparison of gene expression levels in TNBC vs. non-TNBC, based on RNAseq data from breast cancer patients. ( e ) TMA IHC staining for total Axl, Met, and MerTK in TNBC and HER2+ breast cancer (lower panel). Scale bars are 0.5 mm for 2.5× and 50 μm for 20×. Violin plots show the quantification of each protein expression based on the H-scoring in TNBC vs. HER2+ (two-tailed t -test). ( f ) The Kaplan–Meier survival estimate for MerTK, Met, and Axl based on the RNAseq data from all breast cancer patients. Abe: abemaciclib. The original western blot figures can be found in File S1.

Article Snippet: The following antibodies were used for immunoblotting: phospho-Met (Tyr1234/1235) (CST, 3077), Met (D1C2) (CST, 8198), Axl (C89E7) (CST, 8661), phospho-Axl (Y779) (R&D Systems, MAB6965), phospho-MerTK (Phosphosolutions, Denver, CO, USA, p186-749), MerTK (Abcam, Cambridge, UK, ab52968), phospho-Akt (CST, 9271), phospho-mTOR (abclonal, AP0094), and ERBB2 (CST, 2165).

Techniques: Western Blot, Comparison, Gene Expression, Immunohistochemistry, Expressing, Two Tailed Test

Journal: Cancers

Article Title: Targeting Tyro3, Axl, and MerTK Receptor Tyrosine Kinases Significantly Sensitizes Triple-Negative Breast Cancer to CDK4/6 Inhibition

doi: 10.3390/cancers16122253

Figure Lengend Snippet: The combination of sitravatinib with abemaciclib or palbociclib is highly toxic against TNBC cells. ( a ) Chemical structure of sitravatinib (Sitra). ( b ) Immunoblot was performed on cell lines treated for 24 h with Abe (2 μm), Palbo (5 μm), and/or Sitra (2 μm). Protein levels were determined for phospho-AXL, phosho-MET, and phosho-MERTK. ( c ) The clonogenic assay showing that the combination of Abe or Palbo with Sitra significantly decreased the colony formation capacity of TNBC cells. Representative images of stained colonies. ( d ) Combination index (CI) values for the combinations of sitravatinib or merestinib with CDK4/6 inhibitor abemaciclib using different doses. Circles represent experimentally determined CI values using the Chou–Talalay method. The colors (orange and blue) represent the fixed ratio mixtures. ( e , f ) Overview of the toxicity and synergy scores of the drug combinations for TNBC lines. The heatmaps show the level of toxicity ( e ) and Bliss number ( f ) for the cell lines tested in this study. Average values of toxicity ( e ) or Bliss number ( f ) for cells treated with sitravatinib (S) at varying doses (S0 = No Drug, S1 = 1 μm, S2 = 2 μm, and S3 = 3 μm) in combination with either abemaciclib (A) at varying doses (A0 = No Drug, A1 = 1 μm, A2 =2 μm, A3 = 3 μm, and A4 = 4 μm) or palbociclib at varying doses (P0 = No Drug, P1 = 1 μm, P2 = 2 μm, P3 = 3 μm, and P4 = 4 μm). ( g ) Shown is the caspase-3/7 activity measured upon 24 h of drug treatments. The data are presented as mean ± SEM from three independent experiments, expressed as ratios to untreated control values, with associated p values as indicated (One-way ANOVA with Dunnett’s multiple comparisons test analysis). Abe: abemaciclib; Palbo: palbociclib. The original western blot figures can be found in File S1.

Article Snippet: The following antibodies were used for immunoblotting: phospho-Met (Tyr1234/1235) (CST, 3077), Met (D1C2) (CST, 8198), Axl (C89E7) (CST, 8661), phospho-Axl (Y779) (R&D Systems, MAB6965), phospho-MerTK (Phosphosolutions, Denver, CO, USA, p186-749), MerTK (Abcam, Cambridge, UK, ab52968), phospho-Akt (CST, 9271), phospho-mTOR (abclonal, AP0094), and ERBB2 (CST, 2165).

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

Journal: Cancers

Article Title: Targeting Tyro3, Axl, and MerTK Receptor Tyrosine Kinases Significantly Sensitizes Triple-Negative Breast Cancer to CDK4/6 Inhibition

doi: 10.3390/cancers16122253

Figure Lengend Snippet: Lapatinib-resistant HER2+ cell lines became more sensitive to the combination of sitravatinib with abemaciclib or palbociclib. ( a ) Overview of the toxicity of the drug combinations for HER2+ cell lines. The heatmaps show the level of toxicity for the cell lines tested. Average values of toxicity for cells treated with sitravatinib (S) at varying doses (S0 = No Drug, S1 = 1 μm) in combination with either abemaciclib (A) (A0 = No Drug, A1 = 1 μm, and A2 = 2 μm) or palbociclib (P0 = No Drug, P1 = 1 μm, and P2 = 2 μm). ( b ) The clonogenic assay showing that the combination of Abe or Palbo with Sitra had only modest effect on the HER2+ cell line SKBR3. Representative images of stained colonies. ( c ) Schematic representation of the generation of lapatinib-resistant (LapR) HER2 lines through continuous lapatinib treatment with gradual increase in treatment dose up to 30 μm. Cell viability confirming the resistance of the LapR cells to high doses of lapatinib (30 μm). ( d , e ) qRT-PCR and immunoblot showing increased expressions of Axl, Met, and MerTK with the suppression of Her2 levels in LapR vs. the parental cells. ( f ) Cell viability showing increased sensitivity of SKBR3 LapR cells to the combination of abemaciclib or palbociclib with sitravatinib compared with the parental SKBR3 cells. Overview of the toxicity of the drug combinations for HER2+ and LapR HER2 cell lines. The heatmaps show the level of toxicity for the cell lines tested. Average values of toxicity for cells treated with sitravatinib (S) at varying doses (S0 = No Drug, S1 = 1 μm, and S2 = 2 μm) in combination with either abemaciclib (A) (A0 = No Drug, A1 = 1 μm, A2 = 2 μm, and A3 = 3 μm) or palbociclib (P0 = No Drug, P1 = 1 μm, and P2 = 2 μm). ( g ) The clonogenic assay showing that SKBR3-LapR cells became highly sensitive to the combination of Abe or Palbo with Sitra. Representative images of stained colonies. Abe: abemaciclib; Palbo: palbociclib; Sitra: sitravatinib. Each bar represents mean ± SEM from three independent experiments, with associated p (* p < 0.05, *** p < 0.0001; one-way ANOVA with post hoc Tukey analysis). The original western blot figures can be found in File S1.

Article Snippet: The following antibodies were used for immunoblotting: phospho-Met (Tyr1234/1235) (CST, 3077), Met (D1C2) (CST, 8198), Axl (C89E7) (CST, 8661), phospho-Axl (Y779) (R&D Systems, MAB6965), phospho-MerTK (Phosphosolutions, Denver, CO, USA, p186-749), MerTK (Abcam, Cambridge, UK, ab52968), phospho-Akt (CST, 9271), phospho-mTOR (abclonal, AP0094), and ERBB2 (CST, 2165).

Techniques: Clonogenic Assay, Staining, Quantitative RT-PCR, Western Blot

Journal: Cells

Article Title: Mertk Interacts with Tim-4 to Enhance Tim-4-Mediated Efferocytosis

doi: 10.3390/cells9071625

Figure Lengend Snippet: Tim-4 is colocalized with Mertk. ( A ) Schematic diagram of Mertk and Tim-4. Ig, immunoglobulin; FnIII, fibronectin type-III; PKD, protein tyrosine kinase domain; IgV, Immunoglobulin variable; ECR, extracellular region. ( B ) LR73 cells transfected with the indicated plasmids were incubated with TAMRA-labeled apoptotic thymocytes in serum-free alpha-MEM for 2 h, washed with PBS, trypsinized, and analyzed using flow cytometry. Cells that stained double-positive for TAMRA and GFP were considered to be phagocytes engulfing apoptotic cells ( n = 4). ( C ) LR73 cells transfected with the indicated plasmids were incubated with TAMRA-labeled apoptotic thymocytes in serum-free alpha-MEM at 4 °C for 2 h and washed with PBS to remove unbound apoptotic cells. Bound apoptotic cells were then counted ( n ≥ 50 cells). ( D ) LR73 cells transfected with HA-Tim-4 and Mertk-FLAG were incubated with anti-Mer and anti-HA antibodies, and then stained with Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies. Images were acquired by confocal microscopy ( n = 3). Scale bar, 20 µm. ( E , F ) A proximity ligation assay was performed using LR73 cells transfected with the indicated plasmids. One day after transfection, the cells were fixed, blocked, and incubated with anti-FLAG and anti-Tim-4 tail antibodies. The cells then were incubated with the amplification solution at 37 °C overnight. Images were acquired by confocal microscopy (( E ), n = 3) and quantified (( F ), n ≥ 27 cells). Scale bar, 40 µm. Data are shown as the mean ± standard deviation. NS, not significant. ** p < 0.01. *** p < 0.001.

Article Snippet: The antibodies used in this study were anti-FLAG (F1804, Sigma Aldrich, St. Louis, MO, USA), anti-HA (SC-7392, Santa Cruz biotechnology, Dallas, TX, USA), anti-HA (#3724, Cell signaling technology, Danvers, MA, USA), anti-GFP (ab290, Abcam, Cambridge, MA, USA), anti-GST (SC-138, Santa Cruz biotechnology, Dallas, TX, USA), anti-mouse Mertk (AF591, R&D Systems, Minneapolis, MN, USA), anti-Tim-4 (SC-79143, Santa Cruz biotechnology, Dallas, TX, USA), anti-Tim-4 (ab176486, Abcam, Cambridge, MA, USA), anti-Actin (SC-47778, Santa Cruz biotechnology, Dallas, TX, USA), and normal goat IgG control (AB-108-C, R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Incubation, Labeling, Flow Cytometry, Staining, Confocal Microscopy, Proximity Ligation Assay, Amplification, Standard Deviation

Journal: Cells

Article Title: Mertk Interacts with Tim-4 to Enhance Tim-4-Mediated Efferocytosis

doi: 10.3390/cells9071625

Figure Lengend Snippet: Tim-4 interacts with Mertk. ( A ) 293T cells were transfected with the indicated plasmids. Two days after transfection, the cells were lysed, and FLAG-tagged Mertk was precipitated with anti-FLAG antibody-conjugated agarose beads. Bound proteins were detected with the indicated antibodies ( n = 7). ( B ) The lysates of 293T cells transfected with the indicated plasmids were incubated with an anti-Tim-4 tail antibody and protein A/G agarose beads. Bound proteins were detected by immunoblotting ( n = 3). ( C ) The lysates of peritoneal macrophages were incubated with an anti-Tim-4 tail antibody or a control antibody and protein A/G agarose beads. Co-precipitated Mertk was detected by immunoblotting ( n = 3). ( D , E ) 293T cells were transfected with the indicated plasmids and lysed. Mertk-FLAG (( D ), n = 3) or HA-Tim-4 (( E ), n = 3) in the lysates was precipitated with the indicated antibodies, and bound proteins were detected by immunoblotting. TCL, total cell lysate; IP, immunoprecipitation.

Article Snippet: The antibodies used in this study were anti-FLAG (F1804, Sigma Aldrich, St. Louis, MO, USA), anti-HA (SC-7392, Santa Cruz biotechnology, Dallas, TX, USA), anti-HA (#3724, Cell signaling technology, Danvers, MA, USA), anti-GFP (ab290, Abcam, Cambridge, MA, USA), anti-GST (SC-138, Santa Cruz biotechnology, Dallas, TX, USA), anti-mouse Mertk (AF591, R&D Systems, Minneapolis, MN, USA), anti-Tim-4 (SC-79143, Santa Cruz biotechnology, Dallas, TX, USA), anti-Tim-4 (ab176486, Abcam, Cambridge, MA, USA), anti-Actin (SC-47778, Santa Cruz biotechnology, Dallas, TX, USA), and normal goat IgG control (AB-108-C, R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Incubation, Western Blot, Control, Immunoprecipitation

Journal: Cells

Article Title: Mertk Interacts with Tim-4 to Enhance Tim-4-Mediated Efferocytosis

doi: 10.3390/cells9071625

Figure Lengend Snippet: The IgV domain of Tim-4 binds to the fibronectin type III domain of Mertk. ( A – C ) 293T cells transfected with the indicated plasmids were lysed and incubated with anti-FLAG antibody-conjugated agarose beads. Bound proteins were detected by immunoblotting (( A ), n = 3; ( B ), n = 3; ( C ), n = 3). ( D ) The lysates of 293T cells transfected with the indicated plasmids were incubated with an anti-GFP antibody and protein A/G agarose beads. Bound proteins were detected by immunoblotting ( n = 3). ( E ) 293T cells transfected with the indicated plasmids were lysed and incubated with anti-FLAG-conjugated agarose beads. Bound proteins were detected by immunoblotting. Noticeably, the IgV domain of Tim-4 was only detected in immunoprecipitants but not in total cell lysates ( n = 2). ( F ) 293T cells transfected with the indicated plasmids were incubated with glutathione-sepharose beads. Bound proteins were detected by immunoblotting ( n = 3). TCL, total cell lysate; IP, immunoprecipitation.

Article Snippet: The antibodies used in this study were anti-FLAG (F1804, Sigma Aldrich, St. Louis, MO, USA), anti-HA (SC-7392, Santa Cruz biotechnology, Dallas, TX, USA), anti-HA (#3724, Cell signaling technology, Danvers, MA, USA), anti-GFP (ab290, Abcam, Cambridge, MA, USA), anti-GST (SC-138, Santa Cruz biotechnology, Dallas, TX, USA), anti-mouse Mertk (AF591, R&D Systems, Minneapolis, MN, USA), anti-Tim-4 (SC-79143, Santa Cruz biotechnology, Dallas, TX, USA), anti-Tim-4 (ab176486, Abcam, Cambridge, MA, USA), anti-Actin (SC-47778, Santa Cruz biotechnology, Dallas, TX, USA), and normal goat IgG control (AB-108-C, R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Incubation, Western Blot, Immunoprecipitation

Journal: Cells

Article Title: Mertk Interacts with Tim-4 to Enhance Tim-4-Mediated Efferocytosis

doi: 10.3390/cells9071625

Figure Lengend Snippet: Disrupting interaction of Tim-4 with Mertk abolishes the synergistic effect of Mertk on Tim-4-mediated efferocytosis. ( A ) The lysates of 293T cells transfected with the indicated plasmids were incubated with anti-FLAG antibody-conjugated agarose beads in the presence of purified GST-Mertk FnIII or GST. Bound proteins on the beads were detected by immunoblotting ( n = 3). ( B , C ) LR73 cells were transfected with HA-Tim-4 and Mertk-FLAG. One day after transfection, the cells were incubated with purified GST-Mertk FnIII or GST for 2 h, stained with anti-Mer and anti-HA antibodies, and labeled with Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary antibodies. Images were acquired by confocal microscopy (( B ), n = 2) and bound apoptotic cells were counted (( C ), n ≥ 21 cells). Scale bar, 20 µm. ( D ) LR73 cells transfected with the indicated plasmids were incubated with TAMRA-labeled apoptotic thymocytes in serum-free alpha-MEM for 2 h in the presence of purified GST-Mertk FnIII or GST, washed with PBS, trypsinized, and analyzed using flow cytometry ( n = 4). ( E ) LR73 cells were transfected with HA-Tim-4 and Mertk-FLAG. One day after transfection, the cells were incubated with TAMRA-labeled apoptotic thymocytes in serum-free alpha-MEM at 4 °C for 2 h, washed, fixed, stained with anti-Mer and anti-HA antibodies, and labeled with Alexa Fluor 488- and Alexa Fluor 405-conjugated secondary antibodies ( n ≥ 100 cells). Arrowheads indicate bound apoptotic cells. AC, apoptotic cells. Scale bar, 20 µm. ( F ) Peritoneal macrophages derived from WT , Tim-4 -/- , or Mertk -/- were incubated with TAMRA-labeled apoptotic thymocytes in serum-free RPMI for 2 h in the presence of purified GST-Mertk FnIII or GST, and then peritoneal macrophages engulfing apoptotic thymocytes were analyzed using flow cytometry ( n = 4). Data are shown as the mean ± standard deviation. NS, not significant. ** p < 0.01, * p < 0.05. TCL, total cell lysate; IP, immunoprecipitation.

Article Snippet: The antibodies used in this study were anti-FLAG (F1804, Sigma Aldrich, St. Louis, MO, USA), anti-HA (SC-7392, Santa Cruz biotechnology, Dallas, TX, USA), anti-HA (#3724, Cell signaling technology, Danvers, MA, USA), anti-GFP (ab290, Abcam, Cambridge, MA, USA), anti-GST (SC-138, Santa Cruz biotechnology, Dallas, TX, USA), anti-mouse Mertk (AF591, R&D Systems, Minneapolis, MN, USA), anti-Tim-4 (SC-79143, Santa Cruz biotechnology, Dallas, TX, USA), anti-Tim-4 (ab176486, Abcam, Cambridge, MA, USA), anti-Actin (SC-47778, Santa Cruz biotechnology, Dallas, TX, USA), and normal goat IgG control (AB-108-C, R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Incubation, Purification, Western Blot, Staining, Labeling, Confocal Microscopy, Flow Cytometry, Derivative Assay, Standard Deviation, Immunoprecipitation

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: 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