Journal: Journal of Inflammation (London, England)

Article Title: The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization

doi: 10.1186/s12950-015-0081-4

Figure Lengend Snippet: GW9662 induces M2c-like cells that upregulate MerTK and its ligand Gas6. (A-C) Healthy monocytes were cultured in serum-free medium in the absence of cytokines or growth factors (M0 differentiation), with or without the PPAR-γ antagonist GW9662 (2.5-10 μM), for 4 days; when specified, the PPAR-γ agonist rosiglitazone (1 μM) was added. Expression of MerTK, CD163 and CD16 was measured by flow cytometry. (D-E) Gas6 production levels were quantified by ELISA in culture medium, upon incubation with or without GW9662 (2.5-10 μM) of otherwise untreated cells (M0 conditions), LPS (50 ng/ml; M1 conditions) or IL-4 (20 ng/ml; M2a conditions) exposed cells. (A-E) Pooled data are represented as mean values ± SEM. Analysis was performed using one-way repeated measures ANOVA with Newman-Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. When not specified by additional graphic signs, statistical annotations (asterisks) refer to comparisons with respect to the relative GW9662 untreated control group. Each set of data is representative of three independent experiments.

Article Snippet: Biotinylated goat polyclonal anti-human Gas6 antibody (R&D Systems), followed by HRP-conjugated streptavidin (Biolegend), was used for detection.

Techniques: Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: Molecular cancer research : MCR

Article Title: GAS6/AXL Inhibition Enhances Ovarian Cancer Sensitivity to Chemotherapy and PARP Inhibition through Increased DNA Damage and Enhanced Replication Stress.

doi: 10.1158/1541-7786.MCR-21-0302

Figure Lengend Snippet: Figure 1. AXL and GAS6 expression is associated with neoadjuvant chemoresponse and survival. A, Percent of AXL staining in tumor samples with good and poor chemoresponse after neoadjuvant chemotherapy. Representative images of AXL staining in tumors before neoadjuvant chemotherapy and at time of interval cytoreductive surgery at 20X magnification. Horizontal lines indicate mean and inter-quartile range. *, P<0.05. B, Mean chemoresponse scores for patients with high and low serum and tumor GAS6 concentration. Chemoresponse score was a composite of radiologic, surgical, and pathologic response at time of interval cytoreductive surgery. Representative images of GAS6 staining in tumors before neoadjuvant chemotherapy at 40X magnification C, Kaplan Meier curve evaluating progression-free survival (PFS, top) and overall survival (OS, bottom) in patients stratified by serum (left) or tumor (right) GAS6 concentration.

Article Snippet: Briefly, the plate was coated with 1 μg/mL AVB-500 in PBS and GAS6 was detected on a Bio Tek 2 plate reader with a biotinylated polyclonal anti-human GAS6 antibody (BAF885, R&D Systems) and streptavidin-conjugated to horseradish peroxidase (DY998, R&D systems).

Techniques: Expressing, Staining, Concentration Assay

Journal: Molecular cancer research : MCR

Article Title: GAS6/AXL Inhibition Enhances Ovarian Cancer Sensitivity to Chemotherapy and PARP Inhibition through Increased DNA Damage and Enhanced Replication Stress.

doi: 10.1158/1541-7786.MCR-21-0302

Figure Lengend Snippet: Figure 2. GAS6/AXL inhibitor AVB-500 improves response to carboplatin and paclitaxel in ovarian cancer cell lines. A, Colony formation assay in cells treated with 0.5 μM Carboplatin (Carbo), 1 μM AVB-500, or a combination of carboplatin and AVB-500 (C+A). Top, representative images; bottom, quantitation of percent to control absorbance at 590nm. Cells were treated for 72 hours and then incubated in media with 10% FBS until vehicle-treated cells formed colonies optimal for visualization. B, Colony formation assay in cells treated with 50 nM Paclitaxel, 1 μM AVB-500, or a combination of paclitaxel and AVB-500 (P+A). Cells were treated for 72 hours and then incubated in media with 10% FBS until vehicle-treated cells formed colonies optimal for visualization. C, Loewe synergism analysis of OVCAR8 and OVCAR3-TPMES cells treated with varying doses of carboplatin and AVB-500.

Article Snippet: Briefly, the plate was coated with 1 μg/mL AVB-500 in PBS and GAS6 was detected on a Bio Tek 2 plate reader with a biotinylated polyclonal anti-human GAS6 antibody (BAF885, R&D Systems) and streptavidin-conjugated to horseradish peroxidase (DY998, R&D systems).

Techniques: Colony Assay, Quantitation Assay, Control, Incubation

Journal: Molecular cancer research : MCR

Article Title: GAS6/AXL Inhibition Enhances Ovarian Cancer Sensitivity to Chemotherapy and PARP Inhibition through Increased DNA Damage and Enhanced Replication Stress.

doi: 10.1158/1541-7786.MCR-21-0302

Figure Lengend Snippet: Figure 3. GAS6/AXL inhibitor AVB-500 improves response to carboplatin and decreases tumor burden in mouse models of ovarian cancer. A, Volume of tumors over 12 days of treatment in mice engrafted with OVCAR8 or PB1 cells and treated with vehicle, 80mg/kg IP weekly carboplatin + 20mg/kg paclitaxel IP weekly paclitaxel (CT), 30mg/kg AVB-500, or chemotherapy and AVB-500 (CT+A). B, Tumor burden of mice bearing OVCAR8 or OVCAR5 tumors as quantified by tumor weight and number of tumor nodules after treatment with vehicle, CT, AVB-500, or CT+A. C, Quantitation of GAS6 and pAKT immunohistochemistry staining in OVCAR8 tumors in mice treated with vehicle, CT, AVB-500, or CT+A.

Article Snippet: Briefly, the plate was coated with 1 μg/mL AVB-500 in PBS and GAS6 was detected on a Bio Tek 2 plate reader with a biotinylated polyclonal anti-human GAS6 antibody (BAF885, R&D Systems) and streptavidin-conjugated to horseradish peroxidase (DY998, R&D systems).

Techniques: Quantitation Assay, Immunohistochemistry, Staining

Journal: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 2. Regulation of Axl shedding. L929 (A) and L929R (B) cells were treated with 200 ng/ml of PMA for different times, and the concentration of sAxl in cell-conditioned medium was evaluated by a specific ELISA. Untreated cells were used as controls. *, P 0.05 versus control samples. L929 (C) and L929R (D) cells were left untreated or treated with PMA for 2 h. Expression of membrane-bound Axl was evaluated by flow cytometry. (E and F) Cells were treated with PMA (200 ng/ml), Gas6 (50 ng/ml), IL-15 (50 ng/ml), TNF- (10 ng/ml), and LPS (10 ng/ml) for 2 h, and the concentration of sAxl in the culture medium was quantified by ELISA. Untreated (medium) cells were used as controls. **, P 0.01 versus control samples.

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Expressing, Membrane, Cytometry

Journal: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 7. sAxl is present in mouse serum and associates with Gas6. (A) Sera from C57BL/6, CH3, BALB/c, Axl/, Axl/, and Axl/

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 8. Immobilized Axl-Fc chimeric protein promotes cell migra- tion and induces the phosphorylation of Axl and PI3K. (A) L929R cells were plated onto Axl-Fc- or IL-3R–Fc (control)-coated six-well plates and allowed to grow in the presence of 10% FCS. After 18 h, a wound was created by scratching with a pipette tip (0 h). The cells were washed with PBS and incubated further to allow migration into the wounded area. Phase-contrast images of matched pairs of marked wound regions were taken 6 and 18 h later to assess cell migration. (B) L929R cells were serum starved for 4 h and incubated in Axl-Fc- coated wells for 15 min. Incubation in IL-3R–Fc (control)-coated wells or stimulation with 100 ng/ml of Gas6 was used as a negative or positive control, respectively. Cells were lysed, and Axl or PI3K was immunoprecipitated from the lysates with specific Abs. Precipitates were subjected to 10% SDS-PAGE and analyzed with anti-pTyr Abs. Detection of Axl or PI3K on the same blots was used as a loading control. The picture is representative of three independent experi- ments, all of which yielded highly comparable results. IP, immunopre- cipitation; WB, Western blotting.

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques: Phospho-proteomics, Control, Transferring, Incubation, Migration, Positive Control, Immunoprecipitation, SDS Page, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 9. Immobilized Axl-Fc chimeric protein induces cell migration and phosphorylation of Axl and PI3K in WT but not Axl/ MEFs. (A) MEFs were plated onto Axl-Fc- or IL-3R–Fc (control)-coated six-well plates. A confluent cell monolayer was wounded by scratching with a pipette tip (0 h). The cells were washed with PBS and incubated further to allow migration into the wounded area. Phase-contrast images of matched pairs of marked wound regions were taken 6 and 18 h later to assess cell migration. (B) MEFs were serum starved for 4 h and incubated in Axl-Fc-coated wells for 15 min. Incubation in IL-3R–Fc-coated (control) wells or stimulation with 100 ng/ml of Gas6 was used as a negative or positive control, respectively. Cells were lysed, and Axl or PI3K was immunoprecipitated from the lysates with specific Abs. Precipitates were subjected to 10% SDS-PAGE and analyzed with anti-pTyr Abs. Detection of Axl or PI3K on the same blots was used as a loading control. IP, immunoprecipitation; WB, Western blotting.

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques: Migration, Phospho-proteomics, Control, Transferring, Incubation, Positive Control, Immunoprecipitation, SDS Page, Western Blot