Journal: Disease Markers

Article Title: Role of Gas6 and TAM Receptors in the Identification of Cardiopulmonary Involvement in Systemic Sclerosis and Scleroderma Spectrum Disorders

doi: 10.1155/2020/2696173

Figure Lengend Snippet: Comparison of Gas6, sAxl and sMer according to cardiopulmonary involvement.

Article Snippet: A 96-well plate (NUNC Products, Thermo Scientific Inc. MA, USA) was coated with the primary antibody, Goat-IgG anti-human Gas6 (R&D Systems, Minneapolis, USA), and left incubated overnight at room temperature.

Techniques: Comparison

Journal: Disease Markers

Article Title: Role of Gas6 and TAM Receptors in the Identification of Cardiopulmonary Involvement in Systemic Sclerosis and Scleroderma Spectrum Disorders

doi: 10.1155/2020/2696173

Figure Lengend Snippet: (a) ROC curve for sMer in diagnosing PAH. Area under the ROC curve (AUC) 0.697, p < 0.03. (b) ROC curve for Gas6 in diagnosing severe ILD. AUC 0.787, p < 0.001. (c) ROC curve for sAxl in diagnosing severe ILD. AUC 0.705, p < 0.05.

Article Snippet: A 96-well plate (NUNC Products, Thermo Scientific Inc. MA, USA) was coated with the primary antibody, Goat-IgG anti-human Gas6 (R&D Systems, Minneapolis, USA), and left incubated overnight at room temperature.

Techniques:

Journal: Cancer Research

Article Title: Sulfasalazine Suppresses Drug Resistance and Invasiveness of Lung Adenocarcinoma Cells Expressing AXL

doi: 10.1158/0008-5472.can-06-3191

Figure Lengend Snippet: Figure 1. In vitro invasion, doxorubicin resistance, and AXL gene expression of CL1 sublines. A, the morphology and immunohistochemical staining patterns of parental CL1-0 cells and the more invasive CL1-5 cells with anti-AXL antibody. Bar, 20 Am. B, cell invasiveness. The invaded cells were counted 24 h after seeding with 5 104 cells onto the upper chamber of the Matrigel invasion chamber. Columns, mean; bars, SE. C, chemosensitivity of the CL1 sublines. The dosage-dependent inhibitory effects of doxorubicin on cell growth were determined by a tetrazolium-based semiautomated colorimetric MTT assay. D, protein levels of the RTKs (AXL, MER, SKY, EGFR, ErbB2, EphA2, and Met) and the ligand, Gas6. Western blot analysis was done on all five CL1 cell sublines.

Article Snippet: Each sample equivalent of 50 Ag total protein was separated by 8% to 12% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, and probed with specific antibodies against AXL ( for the COOH terminus of AXL, a goat polyclonal IgG; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; for the NH2 terminus of AXL, MAB154, a mouse monoclonal IgG; R&D Systems, Inc., Minneapolis, MN), MER (a goat polyclonal IgG; Santa Cruz Biotechnology), SKY (a goat polyclonal IgG; Santa Cruz Biotechnology), Gas6 (a goat polyclonal IgG; Santa Cruz Biotechnology), EGFR (a rabbit polyclonal IgG; Santa Cruz Biotechnology), phosphorylated EGFR (pTyr1086, a mouse monoclonal IgG; Invitrogen, Carlsbad, CA), EphA2 (a rabbit polyclonal IgG; Santa Cruz Biotechnology), ErbB2 (AB1369, a rabbit polyclonal IgG; Chemicon International, Inc., Temecula, CA), phosphorylated ErbB2 (p-ErbB2; pTyr1221/1222, a mouse monoclonal IgG; Cell Signaling Technology, Inc. Danvers, MA), c-Met (a rabbit polyclonal IgG; Santa Cruz Biotechnology), InBa (a rabbit polyclonal IgG; Santa Cruz Biotechnology), NF-nB p65 (a rabbit polyclonal IgG; Santa Cruz Biotechnology), phosphorylated AKT (p-AKT; a rabbit polyclonal IgG; Biosource, Carlsbad, CA), phosphorylated ERK (p-ERK; a mouse monoclonal IgG1; Santa Cruz Biotechnology), ERK1/2 (a goat polyclonal IgG; Santa Cruz Biotechnology), GSK3a/h (a rabbit polyclonal IgG; Cell Signaling, Waltham, MA), h-catenin (a mouse IgG1; BD Biosciences), actin (a rabbit polyclonal IgG; Sigma-Aldrich Chemical), and SP-1 (a rabbit polyclonal IgG1; Novus Biologicals, Littleton, CO).

Techniques: In Vitro, Gene Expression, Immunohistochemical staining, Staining, MTT Assay, Western Blot

Journal: Cancer Research

Article Title: Sulfasalazine Suppresses Drug Resistance and Invasiveness of Lung Adenocarcinoma Cells Expressing AXL

doi: 10.1158/0008-5472.can-06-3191

Figure Lengend Snippet: Figure 4. Overexpression of AXL increased invasiveness through the activation of NF-nB. A, analysis of NF-nB signaling pathway proteins. Nuclear (NF-nB p65) and cytoplasmic (InB) protein extracts were prepared from shAXL-F4-2485 and pAXL-CL1-0 (clone 22) cells, with or without exogenous Gas6 ligand, for Western blot analysis. Actin and transcription factor SP-1 were used as the loading controls for cytoplasmic and nuclear proteins, respectively. B, NF-nB activity levels in various CL1 sublines and transfectants by the reporter assay. Cells were examined by transient transfection with the NF-nB–driven luciferase reporter in the presence of 10 ng/mL 12-O-tetradecanoylphorbol-13-acetate. Columns, mean; bars, SE. *, P < 0.05; **, P < 0.01, compared with mock vector control, Student’s paired t test. C, suppression of cell invasiveness by dominant-negative InB. Stable transfection of the pInBDN vector significantly reduced the cell invasion rate of CL1-5F4 cells. *, P < 0.05, compared with mock vector control, Student’s paired t test. Expression of the dominant-negative InBa protein was verified by Western blot analysis.

Article Snippet: Each sample equivalent of 50 Ag total protein was separated by 8% to 12% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, and probed with specific antibodies against AXL ( for the COOH terminus of AXL, a goat polyclonal IgG; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; for the NH2 terminus of AXL, MAB154, a mouse monoclonal IgG; R&D Systems, Inc., Minneapolis, MN), MER (a goat polyclonal IgG; Santa Cruz Biotechnology), SKY (a goat polyclonal IgG; Santa Cruz Biotechnology), Gas6 (a goat polyclonal IgG; Santa Cruz Biotechnology), EGFR (a rabbit polyclonal IgG; Santa Cruz Biotechnology), phosphorylated EGFR (pTyr1086, a mouse monoclonal IgG; Invitrogen, Carlsbad, CA), EphA2 (a rabbit polyclonal IgG; Santa Cruz Biotechnology), ErbB2 (AB1369, a rabbit polyclonal IgG; Chemicon International, Inc., Temecula, CA), phosphorylated ErbB2 (p-ErbB2; pTyr1221/1222, a mouse monoclonal IgG; Cell Signaling Technology, Inc. Danvers, MA), c-Met (a rabbit polyclonal IgG; Santa Cruz Biotechnology), InBa (a rabbit polyclonal IgG; Santa Cruz Biotechnology), NF-nB p65 (a rabbit polyclonal IgG; Santa Cruz Biotechnology), phosphorylated AKT (p-AKT; a rabbit polyclonal IgG; Biosource, Carlsbad, CA), phosphorylated ERK (p-ERK; a mouse monoclonal IgG1; Santa Cruz Biotechnology), ERK1/2 (a goat polyclonal IgG; Santa Cruz Biotechnology), GSK3a/h (a rabbit polyclonal IgG; Cell Signaling, Waltham, MA), h-catenin (a mouse IgG1; BD Biosciences), actin (a rabbit polyclonal IgG; Sigma-Aldrich Chemical), and SP-1 (a rabbit polyclonal IgG1; Novus Biologicals, Littleton, CO).

Techniques: Over Expression, Activation Assay, Western Blot, Activity Assay, Reporter Assay, Transfection, Luciferase, Plasmid Preparation, Control, Dominant Negative Mutation, Stable Transfection, Expressing

Journal: Oncotarget

Article Title: Novel Axl-driven signaling pathway and molecular signature characterize high-grade ovarian cancer patients with poor clinical outcome.

doi: 10.18632/oncotarget.5087

Figure Lengend Snippet: Figure 1: Gas6 is expressed in EOC cells and activates the TAM RTK Axl. A. Real-time PCR showing the levels of mRNA for Gas6, AXL, MERTK, and TYRO-3 in six EOC cell lines. Results are presented as relative expression normalized to GAPDH mRNA levels. B. Western blotting on the total cell lysates from the same six EOC cell lines. C. IP with Anti-P-Tyrosine (P-Tyr) performed on lysates from starved or Gas6-stimulated SKOV3 and NL3507 cells. Immunoprecipitated samples were analyzed by western blotting with Abs against the proteins reported on the right. D. Western blotting on the total cell lysates from starved SKOV3 and NL3507 cells pre-treated with Axl-Fc (2.5 μg/ml) and stimulated or not with Gas6 (500 ng/ml). Abs are reported on the right. β-actin was used as the gel loading control.

Article Snippet: Briefly, a 96-well plate (ImmunoPlates MaxiSorp F96, NUNC, Hereford, UK) was coated overnight with anti-Gas6 primary antibody (goat polyclonal affinity purified IgG, R&D Systems).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunoprecipitation, Control

Journal: Oncotarget

Article Title: Novel Axl-driven signaling pathway and molecular signature characterize high-grade ovarian cancer patients with poor clinical outcome.

doi: 10.18632/oncotarget.5087

Figure Lengend Snippet: Figure 2: Gas6-stimulated promotion of invasion through the interaction between ovarian cancer cells and ECM. A. Invasion assay of starved or Gas6-stimulated SKOV3 and NL3507 cells grown in Matrigel. Representative images of three independent experiments are shown. B. Left panel, phase contrast microscopy of starved or Gas6-stimulated SKOV3 cells grown and stimulated with Gas6. C. Upper panel, western blotting on the total cell lysates from starved or Gas6-stimulated SKOV3 and NL3507 cells seeded on FN for 30 min. A representative experiment of the three performed is shown. Abs are reported on the right. β-actin was used as the gel loading control. Lower panel, quantitative evaluation of phosphorylated Axl in Gas6-stimulated cells upon adhesion on FN. The graph reports the mean ± SD from the three independent experiments. D. Upper panel, membrane staining of the integrin β3 receptor determined by flow cytometry on the NL3507 cell line. The expression of the other integrin β receptors on both the SKOV3 and NL3505 cells was also evaluated and the results are reported in Supplementary Fig. 3. The red and light blue peaks, respectively, represent the fluorescence of the cells incubated with the secondary antibody alone as control (α-mouse) and the anti-integrin β3 Ab (Anti-β3). Lower panel, western blotting on the total cell lysates from starved or Gas6-stimulated NL3507 cells, in the absence or in the presence of the anti-integrin β3 Ab. A representative experiment is shown. Abs are reported on the right. β-actin was used as the gel loading control.

Article Snippet: Briefly, a 96-well plate (ImmunoPlates MaxiSorp F96, NUNC, Hereford, UK) was coated overnight with anti-Gas6 primary antibody (goat polyclonal affinity purified IgG, R&D Systems).

Techniques: Invasion Assay, Microscopy, Western Blot, Control, Membrane, Staining, Flow Cytometry, Expressing, Fluorescence, Incubation

Journal: Oncotarget

Article Title: Novel Axl-driven signaling pathway and molecular signature characterize high-grade ovarian cancer patients with poor clinical outcome.

doi: 10.18632/oncotarget.5087

Figure Lengend Snippet: Figure 3: Gas6/Axl signaling triggers PI3K/AKT/rac activation and requires the scaffold protein p130Cas. A. IF performed on SKOV3 cells grown on FN, starved and then induced to migrate through a wound in the presence or not of Gas6. Cells were treated with the reported inhibitors, and F-actin was stained with phalloidin (red). B. Invasion assay of starved or Gas6-stimulated SKOV3 and NL3507 cells grown in Matrigel. Cells were treated with the reported inhibitors. Representative images of the three independent experiments are shown. C. IP performed with anti-p130Cas (upper panel) or with anti-FAK (lower panel), respectively, on lysates of starved or Gas6-stimulated SKOV3 cells. Normal mouse or rabbit (IgG) sera were used as the negative control, respectively. The immunoprecipitated samples were analyzed by western blotting. D. Western blotting on the total cell lysates from SKOV3 cells transiently transfected with a control siRNA (-) or with a pool of siRNA against p130Cas (sip130Cas). SiRNA-transfected SKOV3 cells were starved or Gas6-stimulated and seeded on FN for 30 min. Immunoblottings were performed with Abs against the proteins reported on the right. Alpha (tubulin) was used as the gel loading control.

Article Snippet: Briefly, a 96-well plate (ImmunoPlates MaxiSorp F96, NUNC, Hereford, UK) was coated overnight with anti-Gas6 primary antibody (goat polyclonal affinity purified IgG, R&D Systems).

Techniques: Activation Assay, Staining, Invasion Assay, Negative Control, Immunoprecipitation, Western Blot, Transfection, Control

Journal: Oncotarget

Article Title: Novel Axl-driven signaling pathway and molecular signature characterize high-grade ovarian cancer patients with poor clinical outcome.

doi: 10.18632/oncotarget.5087

Figure Lengend Snippet: Figure 4: Reduction of Gas6-dependent adhesion and invasion following the impairment of p130Cas/Axl interactions. A. Live cell imaging performed on untreated (-) or Gas6-stimulated control siRNA (siCO)- or p130 siRNA (sip130Cas)- transfected SKOV3 cells during adhesion on FN (see Supplementary Video 1 and 2, and Methods). Representative frames at 0, 10 min, and 20 min are reported. Scale bar, 100 μm. B. Graph reporting the number of FN adherent siCo- or sip130Cas-transfected SKOV3 cells, from the live imaging experiment, taken at different time points. Asteriscs indicate the significant differences between the two curves (paired t test, p = 0.0004) C. IF performed on cells as above after 20 min of adhesion on FN. The F-actin was stained with phalloidin (green). D. Invasion assay performed in Matrigel on starved or Gas6-stimulated SKOV3 cells stably p130Cas-silenced (shp130Cas) or infected with an empty shRNA vector (Mock). Representative images of the three independent experiments are shown.

Article Snippet: Briefly, a 96-well plate (ImmunoPlates MaxiSorp F96, NUNC, Hereford, UK) was coated overnight with anti-Gas6 primary antibody (goat polyclonal affinity purified IgG, R&D Systems).

Techniques: Live Cell Imaging, Control, Transfection, Imaging, Staining, Invasion Assay, Stable Transfection, Infection, shRNA, Plasmid Preparation

Journal: Oncotarget

Article Title: Novel Axl-driven signaling pathway and molecular signature characterize high-grade ovarian cancer patients with poor clinical outcome.

doi: 10.18632/oncotarget.5087

Figure Lengend Snippet: Figure 5: In vivo validation on HGEOC samples. A. Gas6 levels measured by ELISA on 22 EOC ascites and 22 non-malignant ascites. B. Evaluation in the EOC ascites of the sequestration by the Axl ectodomain of Gas6. The dosage of the two molecules was performed as described in Methods. The graph reports the molar ratio between Gas6 and Axl ectodomain (sAxl). The red line represents the equimolar ratio. C. Invasion assay performed on starved or Gas6-stimulated MCAs from two representative Axl-positive MCAs from EOC ascites grown in Matrigel. EOC samples containing 80% of HGEOC cells, as evaluated by the cytopathologist, were processed. Satellite cells present in the starved sample are likely to be some immune cells present in the sample D. IP performed on the total cell lysates obtained from the EOC cells of patient #1 with anti-p130Cas. Immunoprecipitated samples were analyzed by western blotting with Abs against the proteins reported on the right. A longer exposure of the immunoblot with both Abs on the total lysates is shown on the right. E. Representative staining with anti-Axl and anti-p130Cas Abs on FFPE sections from two (a and b) HGEOC patients . Scale bar, 50 μm. F. Graph reporting the correlation between the p130Cas and Axl expressions in Type I and Type II (HGEOCs) tumors. The results of the contingency analysis are reported (Fisher’s exact test, p = 0,0009).

Article Snippet: Briefly, a 96-well plate (ImmunoPlates MaxiSorp F96, NUNC, Hereford, UK) was coated overnight with anti-Gas6 primary antibody (goat polyclonal affinity purified IgG, R&D Systems).

Techniques: In Vivo, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Invasion Assay, Immunoprecipitation, Western Blot, Staining

Journal: The Journal of Immunology Author Choice

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: Screening anti-murine MerTK Abs using mouse peritoneal macrophages. (A) Flow cytometric analyses of mouse peritoneal macrophages. (B) Kinetic curve of efferocytosis of pHrodo red–labeled 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 = 3) of three independent experiments, respectively.

Article Snippet: Materials Recombinant murine and human MerTK proteins as well as human Gas6-IgG1-Fc fusion protein were made at Genentech.

Techniques: Labeling, Fluorescence, Positive Control, Control, HTRF Assay

Journal: The Journal of Immunology Author Choice

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: 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 red–labeled 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 macrophage–mediated 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 = 4) of at least two independent experiments.

Article Snippet: Materials Recombinant murine and human MerTK proteins as well as human Gas6-IgG1-Fc fusion protein were made at Genentech.

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