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 pharmacology

Article Title: Pomegranate Peel Extract Decreases Plaque Necrosis and Advanced Atherosclerosis Progression in Apoe -/- Mice.

doi: 10.3389/fphar.2022.888300

Figure Lengend Snippet: FIGURE 4 | (A) TUNEL assay was conducted on aortic root sections from male and female mice treated with PPE or vehicle to determine the number of dead cells in the atherosclerotic plaque. Bar, 50 μm (B) In-situ efferocytosis assay was conducted by immunostaining macrophages with anti-F4/80 antibody (red) and dead cells using TUNEL reagent (green). The number of TUNEL + nuclei that were either associated with an F4/80 + macrophage (white arrow) or were lying free (white arrowheads) was quantified. The associated: free ratio was used as a measure of lesional macrophage efferocytosis efficiency. Bar, 10 µm (C) Immunostaining of aortic root sections with anti-F4/80 and anti-Mertk antibody. The mean fluorescence intensity of the staining was quantified in ImageJ by drawing an ROI around the F4/80 + intimal region of the plaque. Bar, 50 µm. Non-parametric Mann-Whitney U test was conducted to determine statistical significance. *, p < 0.05; **, p < 0.01.

Article Snippet: Biotinylated anti-Mertk antibody (BAF591, R&D Systems) was used as a detection antibody followed by incubation with streptavidin-HRP and Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 8883003 colorimetric development using TMB substrate.

Techniques: TUNEL Assay, In Situ, Immunostaining, Staining, MANN-WHITNEY

Journal: Frontiers in pharmacology

Article Title: Pomegranate Peel Extract Decreases Plaque Necrosis and Advanced Atherosclerosis Progression in Apoe -/- Mice.

doi: 10.3389/fphar.2022.888300

Figure Lengend Snippet: FIGURE 5 | (A) qPCR-based analysis of Mertk mRNA expression in BMDMs treated with vehicle or 50 μg/ml PPE for 16 h n = 3 (B) Flow-cytometry based analysis of cell surface Mertk expression on BMDMs incubated with 50 μg/ml PPE for 16 h followed by treatment with 50 nM PMA for 2 h. The bar graph represents means fluorescence intensity derived from the histogram. n = 4 (C) The levels of soluble Mertk (Sol-Mertk) in the supernatants of BMDMs treated with PMA in the absence or presence of PPE (50 μg/ml) were analyzed by a sandwich ELISA. The bar graph represents the absorbance values measured at 450 nm n = 4 (D) Efferocytosis assay was conducted by incubating fluorescently labeled apoptotic cells (green) with BMDMs treated with PMA (50 nM) in the absence or presence of PPE (50 μg/ml). Bar, 20 μm n = 4 (E) Fluorescence microscopy-based analysis of cytosolic ROS by CellROX staining in BMDMs incubated with vehicle or PPE (50 μg/ml) for 16 h followed by treatment with 50 nM PMA for 2 h. Bar, 20 μm n = 4 (F) Aortic root sections of control or PPE-fed male and female Apoe-/- mice (n = 5 mice per group per sex) were immunostained with anti-8-OHdG to assay lesional ROS-mediated damage. The white dotted line demarcates the intimal region of the plaque. Data were tested for statistical significance using a two-tailed Student’s t-test (A), one-way ANOVA with Sidak’s multiple comparisons test (B–E), or Mann–Whitney test (F). ns, no significant difference; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: Biotinylated anti-Mertk antibody (BAF591, R&D Systems) was used as a detection antibody followed by incubation with streptavidin-HRP and Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 8883003 colorimetric development using TMB substrate.

Techniques: Expressing, Flow Cytometry, Incubation, Derivative Assay, Sandwich ELISA, Labeling, Fluorescence, Microscopy, Staining, Control, Two Tailed Test, MANN-WHITNEY

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

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

doi: 10.4049/jimmunol.2200597

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

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

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

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

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

doi: 10.4049/jimmunol.2200597

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

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

Techniques: Fluorescence, Positive Control, Control, HTRF Assay

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

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

doi: 10.4049/jimmunol.2200597

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

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

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

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

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

doi: 10.4049/jimmunol.2200597

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

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

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

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

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

doi: 10.4049/jimmunol.2200597

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

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

Techniques: Activity Assay

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

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

doi: 10.4049/jimmunol.2200597

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

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

Techniques: In Vitro, Labeling, Control, Activity Assay

Journal: Journal of 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: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Induction of Lysosomal Biogenesis in Atherosclerotic Macrophages Can Rescue Lipid-Induced Lysosomal Dysfunction and Downstream Sequelae

doi: 10.1161/atvbaha.114.303342

Figure Lengend Snippet: Figure 2. Atherosclerotic plaque macrophages display lysosomal dysfunction. A, Fluorescence activated cell sorting (FACS) gating strategy to isolate live CD45+/merTK+/CD64+ resident macro- phages from mouse tissues. B, FACS analysis of spleen, liver, and aortic resident macrophages isolated from pooled tissue (n=3) atherosclerotic (ApoE-/-) mice after 2 months of Western diet and stained with LysoTracker Red (200 nmol/L). C, FACS analysis of aortic resident macrophages isolated from pooled tissue (n=3) wild-type and atherosclerotic (ApoE-/-) mice after 2 months of Western diet and stained with LysoTracker Red. For all bar graphs, mean fluorescence intensity for each peak was determined and expressed as a percentage of Lysotracker stain- ing in splenic macrophages (B) or macrophages from wild-type (nonatherosclerotic) aorta (C). Representative results of ≥3 inde- pendent experiments are shown. FSC indicates forward scatter.

Article Snippet: Single cell suspensions were washed and stained with the following antibodies at 1:200 concentration: CD45 (eBioscience 4805182), CD64 (BD Pharmigen 558539), biotinylated merTK (R&D BAF591), and streptavidin conjugated PerCP-Cy5.5 (BioLegend 405214).

Techniques: Fluorescence, FACS, Isolation, Western Blot, Staining

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: 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: 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