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: 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: The Journal of Clinical Investigation

Article Title: Inflammatory crosstalk impairs phagocytic receptors and aggravates atherosclerosis in clonal hematopoiesis in mice

doi: 10.1172/JCI182939

Figure Lengend Snippet: ( A ) Experimental design. ( B ) Representative H&E staining and quantification of the lesion and necrotic core areas in aortic root sections. Necrotic core regions are indicated by dashed lines. Scale bar: 200 μm. Original magnification, ×100. ( C and D ) Aortic root sections were stained with Masson’s trichrome for the fibrous cap and collagen content area and quantified as the ratio of total lesion area. Scale bar: 100 μm. Original magnification, ×200. ( E and F ) Representative immunofluorescence staining for MERTK ( E ) or TREM2 ( F ) and anti-Mac2 (macrophages). The fluorescence intensity of MERTK ( E ) and TREM2 ( F ) was quantified and normalized by the lesional macrophage area. Scale bars: 50 μm. Original magnification, ×200. Data are presented as the mean ± SEM. n = 20 (control), n = 19 (LAB VF), n = 19 (control/ II1r –/– ), n = 18 (LAB VF/ II1r –/– ) ( B and D ); n = 18 (control), n = 18 (LAB VF), n = 19 (control/ II1r –/– ), n = 16 (LAB VF/ II1r –/– ) ( E ); n = 18 (control), n = 17 (LAB VF), n = 17 ( control/ II1r –/– ), n = 16 (LAB VF/ II1r –/– ) ( F ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-way ANOVA.

Article Snippet: The antibodies used for staining included the following: anti–Mac-2 (Cedarlane, CL8942AP, 1:10,000); GFP (Abcam, ab13970, 1:500); MERTK (R&D Systems, BAF591, 1:200); anti–IL-1β (Abcam, ab9722 1:200); anti-TREM2 (Denali, 4D9 DC1847 or Proteintech 27599-1-AP, 1:100); anti-H3cit (Abcam, ab5103, 1:100); anti–cleaved gasdermin D (Cell Signaling Technology, 10137S, 1:100); anti-GSDMD (Santa Cruz Biotechnology, sc-393581AF647); anti-MPO (R&D Systems, BAF3667, 1:30); anti-decorin (Abcam, ab277636, 1:100); anti–α-SMA (MilliporeSigma, C6198 1:300); anti-PDGFB (Abcam, ab23914, 1:100); anti-PDGFRα (R&D Systems, AF1062, 1:100); and anti–TGF-β1 (Abcam, ab215715, 1:100).

Techniques: Staining, Immunofluorescence, Fluorescence, Control

Journal: The Journal of Clinical Investigation

Article Title: Inflammatory crosstalk impairs phagocytic receptors and aggravates atherosclerosis in clonal hematopoiesis in mice

doi: 10.1172/JCI182939

Figure Lengend Snippet: ( A ) Staining of macrophages, TUNEL and quantification of efferocytosis (ratio of TUNEL + -free cells to macrophage-associated TUNEL + cells) in aortic root sections. Arrowheads show TUNEL + cells. Scale bar: 50 μm. Original magnification, ×200 (enlarged insets). ( B ) Experimental design. ( C ) Representative H&E staining and quantification of the lesion area and necrotic core area in aortic root sections. Necrotic core regions are indicated by dashed lines. Scale bar: 200 μm. Original magnification, ×100 (enlarged insets). ( D ) Aortic root sections were stained with Masson’s trichrome for the fibrous cap and collagen content area and quantified as the ratio of the total lesion area. Scale bar: 100 μm. Original magnification, ×200 (enlarged insets). ( E ) Representative immunofluorescence staining for H3cit, MPO (NETosis), and Mac2 and quantification of the ratio of the double-positive area (NETs) area to the lesion area. Scale bar: 50 μm. Original magnification, ×200. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-way ANOVA. Data are presented as mean ± SEM. n =14 (control), n = 15 (LAB VF) ( A ); n = 20 (control), n = 19 (LAB VF), n = 20 (control/ Mertk CR ), n = 16 (LAB VF/ Mertk CR ) ( C and D ); n = 20 (control), n = 19 (LAB VF), n = 19 (control/ Mertk CR ), n = 16 (LAB VF/ Mertk CR ) ( E ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by unpaired, 2-tailed Student’s t test ( A ) or 2-way ANOVA ( C and E ).

Article Snippet: The antibodies used for staining included the following: anti–Mac-2 (Cedarlane, CL8942AP, 1:10,000); GFP (Abcam, ab13970, 1:500); MERTK (R&D Systems, BAF591, 1:200); anti–IL-1β (Abcam, ab9722 1:200); anti-TREM2 (Denali, 4D9 DC1847 or Proteintech 27599-1-AP, 1:100); anti-H3cit (Abcam, ab5103, 1:100); anti–cleaved gasdermin D (Cell Signaling Technology, 10137S, 1:100); anti-GSDMD (Santa Cruz Biotechnology, sc-393581AF647); anti-MPO (R&D Systems, BAF3667, 1:30); anti-decorin (Abcam, ab277636, 1:100); anti–α-SMA (MilliporeSigma, C6198 1:300); anti-PDGFB (Abcam, ab23914, 1:100); anti-PDGFRα (R&D Systems, AF1062, 1:100); and anti–TGF-β1 (Abcam, ab215715, 1:100).

Techniques: Staining, TUNEL Assay, Immunofluorescence, Control

Journal: The Journal of Clinical Investigation

Article Title: Inflammatory crosstalk impairs phagocytic receptors and aggravates atherosclerosis in clonal hematopoiesis in mice

doi: 10.1172/JCI182939

Figure Lengend Snippet: ( A ) BMDMs from Mx1-Cre (control) and Jak2 VF mice were pretreated or not with IL-1β (25 ng/mL) for 6 hours and then incubated with PKH26-labeled ACs (red) for 45 minutes at a 5:1 AC/macrophage ratio. Phalloidin (green) was used to label F-actin in cells. Bar graph shows quantification of the efferocytosis of macrophages (arrowheads) relative to the percentage of total macrophages. n = 5 independent experiments. ( B ) BMDMs from Mx1-Cre (control [C]) and Jak2 VF (VF) mice were treated with or not with IL-1β (25 ng/mL) for 6 hours, followed by assays for cell-surface MERTK by flow cytometry and soluble MERTK (Sol-MER) levels in culture media by Western blotting (the lanes were run on the same gel but were noncontiguous). n = 4 independent experiments. ( C ) BMDMs were treated or not with the ADAM17 inhibitor TAPI-0 (5 μM) or IL-1β (25 ng/mL) for 6 hours and then assayed for cellular MERTK and soluble MERTK in culture media by Western blotting. n = 6 independent experiments. Data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-way ANOVA.

Article Snippet: The antibodies used for staining included the following: anti–Mac-2 (Cedarlane, CL8942AP, 1:10,000); GFP (Abcam, ab13970, 1:500); MERTK (R&D Systems, BAF591, 1:200); anti–IL-1β (Abcam, ab9722 1:200); anti-TREM2 (Denali, 4D9 DC1847 or Proteintech 27599-1-AP, 1:100); anti-H3cit (Abcam, ab5103, 1:100); anti–cleaved gasdermin D (Cell Signaling Technology, 10137S, 1:100); anti-GSDMD (Santa Cruz Biotechnology, sc-393581AF647); anti-MPO (R&D Systems, BAF3667, 1:30); anti-decorin (Abcam, ab277636, 1:100); anti–α-SMA (MilliporeSigma, C6198 1:300); anti-PDGFB (Abcam, ab23914, 1:100); anti-PDGFRα (R&D Systems, AF1062, 1:100); and anti–TGF-β1 (Abcam, ab215715, 1:100).

Techniques: Control, Incubation, Labeling, Flow Cytometry, Western Blot

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: 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: 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: 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: PLoS ONE

Article Title: Fabricating retinal pigment epithelial cell sheets derived from human induced pluripotent stem cells in an automated closed culture system for regenerative medicine

doi: 10.1371/journal.pone.0212369

Figure Lengend Snippet: hiPS-RPE cell sheets were cultured for 49 days using both machine and manual culture methods. (A–E) Phase-contrast image (top of each figure) and corresponding fluorescence image (bottom of each figure) of vertical sections of machine-cultured hiPS-RPE cell sheets. (A) Immunofluorescence detection of Na/K ATPase, (B) MERTK, (C) Claudin19, (D) RPE65, and (E) PMEL17. (F–J) Phase-contrast image (top) and corresponding fluorescence image (bottom) of vertical sections of manually cultured hiPS-RPE cell sheets. (F) Immunofluorescence detection of Na, K ATPase, (G) MERTK, (H) Claudin19, (I) RPE65, and (J) PMEL17. Nuclei were stained with DAPI. Scale bars: 20 μm.

Article Snippet: Real-time PCR was performed using six RPE-specific gene primers ( RPE65 : Hs01071462_m1, CRALBP : Hs00165632_m1, MERTK : Hs01031979_m1, BEST1 : Hs04397293_m1, Claudin19 : Hs00961709_m1, Claudin11 : Hs00194440_m1), and GAPDH (Hs02786624_g1, Thermo Fisher Scientific) with 40 cycles of 95 °C for 5 s and 60 °C for 30 s.

Techniques: Cell Culture, Fluorescence, Immunofluorescence, Staining