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: Nature Communications

Article Title: Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa

doi: 10.1038/s41467-018-06448-y

Figure Lengend Snippet: Characterisation of RP11 - RPE cells revealed polarity and functional defects. a Schematic of RPE differentiation timeline; b Bright-field images of iPSC-derived RPE: representative examples from at least ten independent experiments, scale bar 100 μm; c Immunostaining for basolateral markers BEST1 and Na + /K + -ATPase: representative images from three independent experiments, scale bar 50 μm; d Correct basolateral distribution of collagen IV (C-IV) and apical MERTK in unaffected control (WT3) but not RP11 RPE cells: representative images from three independent experiments, scale bar 50 μm; e , f ELISA assays for apical and basal secretion of PEDF and VEGF, respectively, in control and RP11 - RPE cells; g Trans-epithelial resistance measurements revealed a significant difference between patient and RP11 - RPE cells; h Reduced phagocytic capacity in RP11 - RPE cells. Statistical significance is calculated for MFI (mean fluorescence intensity) values. e – h Data shown as mean ± SEM, n = 3. Statistical significance of pairwise comparisons is indicated by n.s.: not significant; *** p < 0.001; **** p < 0.0001 (Student’s paired t test). b – h Data obtained from RPE at week 21 of differentiation

Article Snippet: Cells were treated with primary antibodies anti-bestrophin (Abcam, ab2182, 1:300), anti-sodium potassium ATPase (Alexa Fluor® 488 conjugate) (Abcam, ab197713, 1:50), pericentrin (Abcam, ab28144, 1:500), MERTK (Bethyl, A300-222A, 1:200), ARL13B (Proteintech, 17711-1-AP, 1:500), collagen IV (Abcam, ab6586, 1:200), PRPF31 (Abnova, PAB7154, 1:500) and SNRPB monoclonal antibody (Y12) (ThermoFisher, MA5-13449, 1:500), overnight at 4 °C, and with secondary antibodies anti-rabbit FITC (Sigma, T9887, 1:500) or anti-mouse FITC (Jackson Immuno Research, 715-095-151, 1:500) and anti-mouse Cy3 (Jackson Immuno Research, 115-165-003, 1:500) or anti-rabbit Cy3 (Jackson Immuno Research, 111-165-003, 1:500) diluted in PBS for 1 h at room temperature.

Techniques: Functional Assay, Derivative Assay, Immunostaining, Control, Enzyme-linked Immunosorbent Assay, Fluorescence