Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) IPF signaling pathway was one of the increasingly upregulated pathways over time as revealed by IPA analysis. The gene set contributing to this pathway included distinct groups of early and late profibrotic associated genes (marked by black rectangles) that were serially and time dependently upregulated. Timepoint order forced, genes hierarchical clustered. (B- D) Fold change of mRNA expression levels of TAM RTK family, Axl , Mertk and Tyro3 over the course of repair. (E-F) Fold change of expression level of TAM RTK ligands, Gas6 and Pros1 over the course of repair. Data are shown as mean ± SEM of n= 3-7. P ≤ 0.05 (*); P ≤ 0.01 (**); P ≤ 0.001 (***); P ≤ 0.0001 (****) compared with stuffer control.

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) Schematic of the e xperimental setup to study AXL-GAS6/PROS1 interaction using aFRET in mouse lung tissue sections. Lungs were isolated on day 4 post DT. aFRET is a coincidence assay, labels both receptor (AXL) and ligands (GAS6 or PROS1) (see Methods for details). FRET occurs in the range of 1-10 nm. Scheme was created in BioRender. Geillinger-kästle, K. (2025) https://BioRender.com/h45x771 (B) Representative image of AXL-GAS6 interaction. (C) Representative image of AXL-PROS1 interaction. (B-C) Upper figures showed the mean of the donor lifetime (Ƭ) in the absence of acceptor. Lower figures showed the interaction state between AXL-GAS6/ PROS1 for each coincidental region (marked by yellow and black circles). Different coincidental regions possessed different interaction state. (D) Box and whiskers plots showed the heterogeneity of AXL-GAS6/PROS1 interaction states represented as FRET efficiency (Ef%). Ef% below 4% represents the Förster Radius (R 0 at 5.83nm) and not proteins, interactive state as marked by red dashed line. Data are shown as box and whiskers plot of n=5-6. Each dot represents median of Ef% for each mouse. P ≤ 0.05 (*). (E) Correlation plot between Ef% AXL-GAS6 and AXL expression. (F) Correlation plot between Ef% AXL-PROS1 and AXL expression. (G-H) Surface plasmon resonance response curve showing the binding of rhGAS6 and rhPROS1 in different concentration to rhAXL. (I-J) Concentration dependent binding of rhGAS6 and rhPROS1 to rhAXL. Different colored lines in (G-H) correspond to different colored dots in (I-J), which indicate different concentrations of rhGAS6 and rhPROS1. (K-L) GAS6 and PROS1 concentration in human BALF of non- diseased control, ILD/IPF-, and COPD patients. Data are shown as mean ± SEM of n= 4-8. P ≤ 0.01 (**); P ≤ 0.001 (***).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Isolation, Expressing, SPR Assay, Binding Assay, Concentration Assay, Control

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) 7664 is a tissue section from a healthy lung whereas 1015 and 1033 are sections from IPF patients with various advanced degrees of IPF. The panels show representative images mapping the interactive states of GAS6 and PROS1 with AXL (pseudo-colour heat map of (Ef%) on the expression level of GAS6 and PROS1 (fluorescence images in grey scale). The individual violin plots quantify the heterogeneity of interactive states for both ligands and the receptor. The dotted line at 4% Ef represents the Förster Radius (R 0 ). Values below 4% are not representative of ligand-receptor interactions. (B) Global violin plots of all coincident regions (per pixel) of AXL-GAS6 and AXL-PROS1. Each global violin plot represents 2x10 6 data points. To determine the p values for AXL-GAS6 and AXL-PROS1, using non-parametric Mann- Whitney U test, we utilised 1000 randomly generated data points of the 2x10 6 . Healthy lung has similar distribution of AXL-GAS6 and AXL-PROS1 interactions, whereas the two patient samples show, in one case (1015) no interaction of AXL-GAS6 or AXL-PROS1 and another case (1033) a significantly higher distribution of AXL-PROS1 versus AXL-GAS6. The p values between AXL-GAS6 and AXL-PROS1 are 1.9x10 -3 (7664), 2.4x10 -1 (1015) and 8.2x10 - respectively. The dotted line at 4% Ef represents the Förster Radius (R 0 ).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Expressing, Fluorescence, MANN-WHITNEY, Generated

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: AXL is predominantly expressed in basal and aberrant basaloid cells, promoting proliferation via AXL-GAS6 signaling. (A) UMAP plots visualize AXL and its ligands, GAS6 and PROS1 normalized expression in the epithelial cell compartment of an integrated scRNA-seq IPF atlas. (B) Quantification of AXL and its ligands expression in epithelial cells of IPF patients and the non-diseased controls. (C) Expression of AXL and its ligands within the epithelial cells compartment in the lung of PF patients and the non-diseased controls. (D-E) AXL and its ligands expression in SAEC under submerged culture condition. (F) Representative western blot image validating knockout of AXL via CRISPR/Cas9 in 2 different donors. AXL (140 KDa, red bands), loading control ß-Actin (42 KDa, green bands), and M is protein marker. (G) Quantification of AXL expressions of each donor before and after CRISPR/Cas9 mediated knockdown. Experiments were done in duplicate. (H) Proliferation of AXL WT and AXL KD SAEC as assessed by BrdU in all donors 48 hours post seeding. (I) Correlation between AXL expression in (G) and SAEC proliferation in (H). (J) Proliferation of AXL WT and (K) AXL KD SAEC as assessed by BrdU 48 hours post treatment with rhGAS6/ rhPROS1/ combination of both proteins. WT: Wildtype; KD: Knockdown. Data are shown as mean ± SEM of n= 4 – 7. P > 0.05 (ns/ non-significant); P ≤ 0.05 (*); P ≤ 0.001 (***).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Expressing, Western Blot, Knock-Out, CRISPR, Control, Marker, Knockdown

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) SAEC basal cells were allowed to differentiate for 23 days. On day 23 post ALI, 5 ng/ml rhTGF-ß was added to the culture. On day 26 post ALI, rhTGF-ß was added together with rhGAS6, rhPROS1, or a combination of both. Analysis was performed on day 29 post ALI. Scheme was created in BioRender. Geillinger-kästle, K. (2025) https://BioRender.com/k46g355 . (B) GAS6 and (C) PROS1 concentration in the SAEC cell culture supernatant post rhTGF-ß treatment compared to the BSA control. (D) Epithelial barrier integrity as measured by FITC-dextran permeability assays. (E) Fold change of AXL mRNA expression as measured by qPCR. (F) Correlation between slope measured by FITC- dextran permeability assay (D) and ΔCT of AXL (r= -0.9519 R 2 = 0.9062) measured by qPCR (E). Data are shown as mean ± SEM of n= 4 – 5. P > 0.05 (ns/ non-significant); P ≤ 0.05 (*); P ≤ 0.01 (**).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Concentration Assay, Cell Culture, Control, Permeability, Expressing, FITC-Dextran Permeability Assay

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: After injury, the normal repair process involves an initial inflammatory response, followed by AXL- GAS6 interaction, which promotes cell proliferation. Once sufficient proliferation is achieved, AXL-PROS1 binding occurs, halting proliferation and triggering cell differentiation. This balance between proliferation and differentiation ensures proper repair, leading to healthy alveoli. However, dysbalanced AXL signaling due to repeated injury and persistently high TGF- ß levels, can result in aberrant repair and irreversible fibrosis (IPF). This may occur due to high-affinity interaction between AXL and GAS6 prevents PROS1 from effectively binding to AXL. As a result, excessive proliferation occurs with insufficient differentiation, leading to aberrant repair and fibrosis. Scheme was created in BioRender. Geillinger-kästle, K. (2025) https://BioRender.com/q77c168.xss

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Binding Assay, Cell Differentiation

Journal: International journal of molecular sciences

Article Title: Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

doi: 10.3390/ijms25126630

Figure Lengend Snippet: Figure 2. Gas6 and Protein S bioavailabilities peak at different times of the light–dark cycle. Analysis of the mRNA (A,C) and protein (B,D) expression profiles for Gas6 (A,B) and Protein S (C,D) in the RPE/choroid, retina, or IPM for wildtype (wt, blue) and β5−/−mice (β5 ko, pink) at different times of day as indicated. (A) qPCR experiments allowed us to show that Gas6 mRNA expression levels are slightly increased just before (retina) and after (RPE/choroid) the phagocytic peak in wt mice. Gas6 expression levels were lower in the RPE/choroid of β5−/−mice between peak phagocytosis time and 22.00 while levels were unchanged in the retina fraction. (B) Corresponding protein quantification and representative immunoblots in the IPM of fellow eyes showed a decrease at light onset followed by a marked increase at the time of the phagocytic peak in wt animals. In β5−/−mice, expression levels did not vary. (C) Pros1 mRNA expression increases just before and at the time of peak phagocytosis in wt RPE/choroid and retina, respectively. In both tissue samples, a second peak occurs at night offset (retina) of just after (RPE/choroid). The 7.00 and 22.00 RPE/choroid peaks, as well as the phagocytosis retina peak, are lost in β5−/−mice, but median levels are not changed. (D) Corresponding protein quantification and representative immunoblots in the IPM of fellow eyes follows a combination of RPE/choroid and retina gene expression profiles depicted in (C). Results are in arbitrary units (a.u.) as means ± SDs, n = 3–8 independent samples; reference: wildtype sample at 8.00. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; two-way ANOVA with a Sidak post-test comparing wildtype and β5−/−samples at each time-point. Black bars: time-points during which lights were on (8.00–20.00); grey dotted bar, black tick: phagocytosis peak.

Article Snippet: Gas6 recombinant proteins (mouse, 986-GS), as well as anti-mouse antibodies raised in goats against Gas6 (AF986) and MFG-E8 (AF2805), were from R&D Systems (BioTechne, Noyal-Châtillon-sur-Seiche, France).

Techniques: Expressing, Western Blot, Gene Expression

Journal: International journal of molecular sciences

Article Title: Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

doi: 10.3390/ijms25126630

Figure Lengend Snippet: Figure 3. Gas6 is more expressed than Pros1, and ligands are more expressed in the retina than in the RPE/choroid. (A) Gas6 and Protein S (Pros1) mRNA expression profiles in RPE/choroid (green) and retina (orange) fractions of wildtype (wt) mice were compared at different times of day as indicated. Both ligands were more expressed in the retina than in the RPE/choroid. (B) Respective Gas6 (blue) and Pros1 (pink) mRNA expression profiles were compared in both the RPE/choroid and retina fractions of wt mice at different times of day as indicated. In both tissue types, Gas6 was much more expressed than Pros1. (A,B) Results are in arbitrary units (a.u.) as means ± SDs, n = 3–7 independent samples; references: RPE/choroid (A) or Pros1 (B) sample at 8.00. ** p < 0.01, *** p < 0.001, and **** p < 0.0001; two-way ANOVA with a Sidak post-test comparing wildtype and β5−/−samples at each time-point. Black bars: time-points during which lights were on (8.00–20.00); grey dotted bar, black tick: phagocytosis peak. (C) siRNA samples were used to downregulate the endogenous production of each ligand by RPE-J cells. Cells were then subjected to phagocytosis assays for 1.5 and 3 h as indicated. Decrease in Gas6 synthesis (blue bars) leads to diminished binding and internalization of POSs compared to control siRNA (Ctrl, white bars). Blocking the production of Protein S (Pros1, pink/purple bars) only slightly affects binding at 1.5 h. Adding both siRNAs has the same effect than adding the Gas6 siRNA alone. Targeting of both ligands’ production (purple bars) has the same effect as the decrease in Gas6 alone. Results of FITC/DAPI ratios are in arbitrary units (a.u.) expressed as means ± SDs, n = 5–6 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, one-way ANOVA with a Tukey post-test compared to each series corresponding control; reference: total phagocytosis (binding + internalization) for the control condition.

Article Snippet: Gas6 recombinant proteins (mouse, 986-GS), as well as anti-mouse antibodies raised in goats against Gas6 (AF986) and MFG-E8 (AF2805), were from R&D Systems (BioTechne, Noyal-Châtillon-sur-Seiche, France).

Techniques: Expressing, Binding Assay, Control, Blocking Assay

Journal: International journal of molecular sciences

Article Title: Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

doi: 10.3390/ijms25126630

Figure Lengend Snippet: Figure 5. Gas6 and Protein S bind to different amino acids of MerTK Ig-like domains. Mutants target- ing ligand binding sites in MerTK Ig-like domains 1 (pink bars) and 2 (blue bars) were transfected in RPE-J and tested for their influence on POS binding (top left) and internalization (bottom left) when compared to non-mutated MerTK (black bar) with or without the addition of Gas6 and Protein S— alone or in combination—as indicated. The p.Gly122Arg (G122R, light pink bars) mutant significantly increases POS binding in DMEM while addition of Gas6 diminishes binding and addition of Protein S importantly increases internalization compared to the wt construct. Among the neighbor sites p.Thr140Ala (T140A) and p.Phe142Val (F142V), only p.Phe142Val (F142V) shows a slight increase in POS binding in the presence of Gas6. The p.Lys263Ile (K263I) mutant has a negative impact on both binding and internalization of POSs alone, as well as a positive effect on the internalization of POSs with Gas6. The p.Lys269Leu (K269L) mutant has almost no effect besides slightly less binding of POSs alone. When challenged with fluorescent beads (right bar graphs), no difference was observed in this study between the different clones. Results of FITC/DAPI ratios in arbitrary units (a.u.) are expressed as means ± SDs, with n = 4–6 independent experiments (POSs, left) or n = 3–4 independent experiments (beads, right). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; one-way ANOVA with a Tukey post-test compared to each series corresponding wildtype; reference: total phagocytosis (binding + internalization) for the control condition (WT). Significance brackets compare different ligand conditions for a single mutant.

Article Snippet: Gas6 recombinant proteins (mouse, 986-GS), as well as anti-mouse antibodies raised in goats against Gas6 (AF986) and MFG-E8 (AF2805), were from R&D Systems (BioTechne, Noyal-Châtillon-sur-Seiche, France).

Techniques: Ligand Binding Assay, Transfection, Binding Assay, Mutagenesis, Construct, Clone Assay, Control

Journal: Journal of cellular biochemistry

Article Title: Growth Arrest-Specific 6 (GAS6) Promotes Prostate Cancer Survival by G 1 Arrest/S Phase Delay and Inhibition of Apoptotic Pathway During Chemotherapy in Bone Marrow

doi: 10.1002/jcb.25582

Figure Lengend Snippet: (A) GAS6 expression by osteoblasts (red, white arrows) in the bone marrow of the long bones of GAS6 expressing wild-type (GAS6+/+) mice and GAS6 deficient GAS6 knockout (GAS6−/−) mice as detected by immunofluorescence staining. Blue, DAPI nuclear stain. Bar=20μm. (B) Secretion levels of GAS6 protein in the marrow fluid of the GAS6+/+ and GAS6−/− mice as detected by ELISA. (C) The proliferation assays in PCa cells were performed by XTT. mRNA expression levels of (D) PLK1 and (E) STK15 were measured as qPCR. Data in Fig. 1B–E are representative of mean with s.d. (Student’s t-test).

Article Snippet: PCa cells (PC3 or DU145) (3 x 10 3 ) were seeded onto 96-well culture plates with RPMI 1640, 1% FBS, and 1% P/S and then the cells were treated with human recombinant GAS6 (0–3μg/ml (cat. 885-GSB-050, R&D Systems, Minneapolis, MN) for 3 days.

Techniques: Expressing, Knock-Out, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of cellular biochemistry

Article Title: Growth Arrest-Specific 6 (GAS6) Promotes Prostate Cancer Survival by G 1 Arrest/S Phase Delay and Inhibition of Apoptotic Pathway During Chemotherapy in Bone Marrow

doi: 10.1002/jcb.25582

Figure Lengend Snippet: (A) A flow profile of the cell cycle status; G1, S, and G2/M in Fucci-PC3 cells. (B) % G1 cells in Fucci-PC3 cells following treatment of GAS6 (0–2μg/ml) in standard culture conditions for 72 hours. (C) The living cell imaging of Fucci-PC3 (red arrows) following GAS6 (2μg/ml) treatment by video for 24 hours with 15 min interval by Deltavision Elite Microscope. Bar=20μm. (D) Quantification of G1 duration in the single cell from Fig. 2C (n=10/group). (E) % G1 cells in Fucci-PC3 cell culture following the treatment of docetaxel with the presence or absence of GAS6. (F) % G1 cells in cocultured Fucci-PC3 cells with GAS6 expressing wild-type osteoblasts (GAS6+/+ OB) or GAS6 deficient osteoblasts (GAS−/− OB) following the treatment of docetaxel. Data in Fig. 2B, D–F are representative of mean with s.d. (Student’s t-test).

Article Snippet: PCa cells (PC3 or DU145) (3 x 10 3 ) were seeded onto 96-well culture plates with RPMI 1640, 1% FBS, and 1% P/S and then the cells were treated with human recombinant GAS6 (0–3μg/ml (cat. 885-GSB-050, R&D Systems, Minneapolis, MN) for 3 days.

Techniques: Imaging, Microscopy, Cell Culture, Expressing

Journal: Journal of cellular biochemistry

Article Title: Growth Arrest-Specific 6 (GAS6) Promotes Prostate Cancer Survival by G 1 Arrest/S Phase Delay and Inhibition of Apoptotic Pathway During Chemotherapy in Bone Marrow

doi: 10.1002/jcb.25582

Figure Lengend Snippet: Examination of expression of the checkpoint and cell cycle related proteins; p-CHK2, CHK2 (checkpoint), Cyclin B1 (G2/M marker), Cyclin D1, CDK4, p27, and p21 (G0/G1 marker), and CDC25A, Cyclin E1, CDK2 (S phase entry marker) in the cultures of (A) PC3 cells and (B) DU145 cells following docetaxel treatment with the presence or absence of GAS6 by Western blots.

Article Snippet: PCa cells (PC3 or DU145) (3 x 10 3 ) were seeded onto 96-well culture plates with RPMI 1640, 1% FBS, and 1% P/S and then the cells were treated with human recombinant GAS6 (0–3μg/ml (cat. 885-GSB-050, R&D Systems, Minneapolis, MN) for 3 days.

Techniques: Expressing, Marker, Western Blot

Journal: Journal of cellular biochemistry

Article Title: Growth Arrest-Specific 6 (GAS6) Promotes Prostate Cancer Survival by G 1 Arrest/S Phase Delay and Inhibition of Apoptotic Pathway During Chemotherapy in Bone Marrow

doi: 10.1002/jcb.25582

Figure Lengend Snippet: Examination of % apoptosis in (A) PC3 and (B) DU145 cells following the docetaxel treatment with the presence or absence of GAS6 by FACS analysis using Annexin V staining. Examination of % apoptosis in (C) PC3 and (D) DU145 cells in cocultured PC3 cells with GAS6+/+ OB or GAS−/− OB following the treatment of docetaxel. (E) Apoptotic tumor cells (red, white arrows) in the tumor established PC3 cells within GAS6+/+ vossicles or GAS6−/− vossicles as evaluated by TUNEL staining. Blue, DAPI nuclear stain. Bar=50μm. (F) Quantification of % apoptotic cells from Fig. 4E. Docetaxel-induced apoptotic signaling, Caspases-3 and PARP in (G) PC3 cells and (H) DU145 cells following the treatment of docetaxel with the presence or absence of GAS6 were evaluated by Western blots. Data in Fig. 4A–D, F are representative of mean with s.d. (Student’s t-test).

Article Snippet: PCa cells (PC3 or DU145) (3 x 10 3 ) were seeded onto 96-well culture plates with RPMI 1640, 1% FBS, and 1% P/S and then the cells were treated with human recombinant GAS6 (0–3μg/ml (cat. 885-GSB-050, R&D Systems, Minneapolis, MN) for 3 days.

Techniques: Staining, TUNEL Assay, Western Blot

Journal: Journal of cellular biochemistry

Article Title: Growth Arrest-Specific 6 (GAS6) Promotes Prostate Cancer Survival by G 1 Arrest/S Phase Delay and Inhibition of Apoptotic Pathway During Chemotherapy in Bone Marrow

doi: 10.1002/jcb.25582

Figure Lengend Snippet: GAS6 promotes PCa survival by G1 cell cycle arrest/ S phase delay and inhibition of apoptotic signaling pathways during chemotherapy in bone marrow, which may have important implications for targeting metastatic disease.

Article Snippet: PCa cells (PC3 or DU145) (3 x 10 3 ) were seeded onto 96-well culture plates with RPMI 1640, 1% FBS, and 1% P/S and then the cells were treated with human recombinant GAS6 (0–3μg/ml (cat. 885-GSB-050, R&D Systems, Minneapolis, MN) for 3 days.

Techniques: Inhibition, Protein-Protein interactions

Journal: PLoS ONE

Article Title: Plasma Concentrations Predict Aortic Expression of Growth-Arrest-Specific Protein 6 in Patients Undergoing Coronary Artery Bypass Grafting

doi: 10.1371/journal.pone.0079452

Figure Lengend Snippet: Anthropometric and biochemical variables among CABG patients and matched control subjects.

Article Snippet: Gas6 level was measured by sandwich ELISA with a polyclonal mouse antihuman Gas6 antibody (R&D Systems Europe, Lille, France) as a catcher and a biotinylated goat antiserum as a detector (R&D Systems Europe), as described in our previous study .

Techniques: Control

Journal: PLoS ONE

Article Title: Plasma Concentrations Predict Aortic Expression of Growth-Arrest-Specific Protein 6 in Patients Undergoing Coronary Artery Bypass Grafting

doi: 10.1371/journal.pone.0079452

Figure Lengend Snippet: The lines represent the mean values in each group. The CABG group had significantly lower plasma Gas6 and sAxl levels than the control subject group ( P <0.0001 and P <0.001, respectively).

Article Snippet: Gas6 level was measured by sandwich ELISA with a polyclonal mouse antihuman Gas6 antibody (R&D Systems Europe, Lille, France) as a catcher and a biotinylated goat antiserum as a detector (R&D Systems Europe), as described in our previous study .

Techniques: Clinical Proteomics, Control

Journal: PLoS ONE

Article Title: Plasma Concentrations Predict Aortic Expression of Growth-Arrest-Specific Protein 6 in Patients Undergoing Coronary Artery Bypass Grafting

doi: 10.1371/journal.pone.0079452

Figure Lengend Snippet: Age-adjusted Spearman partial correlation coefficients between Gas6 levels and biochemical variables in CABG and control groups.

Article Snippet: Gas6 level was measured by sandwich ELISA with a polyclonal mouse antihuman Gas6 antibody (R&D Systems Europe, Lille, France) as a catcher and a biotinylated goat antiserum as a detector (R&D Systems Europe), as described in our previous study .

Techniques: Control

Journal: PLoS ONE

Article Title: Plasma Concentrations Predict Aortic Expression of Growth-Arrest-Specific Protein 6 in Patients Undergoing Coronary Artery Bypass Grafting

doi: 10.1371/journal.pone.0079452

Figure Lengend Snippet: Case 1 showed high expression of Axl (A) and Gas6 (B), and case 2 represented low expression of Axl (C) and Gas6 (D). Isotype antibodies for Axl (E) and Gas6 (F) showed negative staining. Similarly, case 1 revealed increased Axl and Gas6 expression compared with that in case 2 in the mRNA (G), real-time polymerase chain reaction (H), and western blotting (I).

Article Snippet: Gas6 level was measured by sandwich ELISA with a polyclonal mouse antihuman Gas6 antibody (R&D Systems Europe, Lille, France) as a catcher and a biotinylated goat antiserum as a detector (R&D Systems Europe), as described in our previous study .

Techniques: Expressing, Negative Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Plasma Concentrations Predict Aortic Expression of Growth-Arrest-Specific Protein 6 in Patients Undergoing Coronary Artery Bypass Grafting

doi: 10.1371/journal.pone.0079452

Figure Lengend Snippet: The positive correlations between plasma sAxl and Gas6 levels were significant in both groups (A, B). The expression of Gas6 was significantly and positively correlated with Axl protein expression in the aorta ( P = 0.0002).

Article Snippet: Gas6 level was measured by sandwich ELISA with a polyclonal mouse antihuman Gas6 antibody (R&D Systems Europe, Lille, France) as a catcher and a biotinylated goat antiserum as a detector (R&D Systems Europe), as described in our previous study .

Techniques: Clinical Proteomics, Expressing

Journal: PLoS ONE

Article Title: Plasma Concentrations Predict Aortic Expression of Growth-Arrest-Specific Protein 6 in Patients Undergoing Coronary Artery Bypass Grafting

doi: 10.1371/journal.pone.0079452

Figure Lengend Snippet: Age-adjusted Spearman partial correlation coefficients between plasma Gas6 levels and tissue expression in CABG group.

Article Snippet: Gas6 level was measured by sandwich ELISA with a polyclonal mouse antihuman Gas6 antibody (R&D Systems Europe, Lille, France) as a catcher and a biotinylated goat antiserum as a detector (R&D Systems Europe), as described in our previous study .

Techniques: Clinical Proteomics, Expressing

Journal: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury.

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: Figure 1: GAS6 and p-AXL are expressed in the regenerative cavernous nerve of rats subjected to BCNI. (A) Representative ICP tracing responses of each group (n=5 per group) to the stimulation of the cavernous nerve at 14 days and 28 days. The green bar represents an electrical stimulus duration of 60 seconds. (B) Immunohistochemistry images demonstrated that GAS6 and p-AXL expression levels increased in the cavernous nerve after BCNI. (C) The quantitative analysis results showed a significantly higher expression of GAS6 and p-AXL in the cavernous nerve of animals subjected to injury after 28 days.

Article Snippet: Primary antibodies against GAS6 (1:100, TA323562; Origene), p-AXL (Y779) (1:100, AF2228, R&D), and CIP2A (1:50, NB100-74663, Novus) were used.

Techniques: Immunohistochemistry, Expressing

Journal: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury.

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: Figure 2: GAS6 induces Schwann cell proliferation. (A) Time- and dose-dependent effects of LDC1267 in RSC96 cells. (B) Time- and dose-dependent effects of AXL-Fc in RSC96 cells. The inhibitory effects of cell proliferation were increased in a time-dependent manner (31.3%, 35.1% and 43.1% decreased in 24 h, 48 h, and 72 h, respectively). Data are the mean ± SD, and n = 3 for each concentration and time point. * p < 0.05, ** p < 0.01, vs. control. (C) RSC96 incubated with GAS6 (100 ng/ml) for 30 min and immunoblotting evaluations of pAxl, Axl, pERK1/2, ERK1/2, pAKT, AKT, Myc and Survivin. (D) RSC96 cells were transfected with either control or Axl siRNA for 48 hours and then exposed to GAS6 (100 ng/ml) for 30 min. The relative quantification of protein expression was normalized with respect to β-actin expression.

Article Snippet: Primary antibodies against GAS6 (1:100, TA323562; Origene), p-AXL (Y779) (1:100, AF2228, R&D), and CIP2A (1:50, NB100-74663, Novus) were used.

Techniques: Concentration Assay, Control, Incubation, Western Blot, Transfection, Quantitative Proteomics, Expressing

Journal: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury.

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: Figure 3: GAS6 increases the amount of both CIP2A and p-DAPK in RSC96 cells. (A) RSC96 cells were incubated with GAS6 (100 ng/ml), and immunoblotting evaluations of CIP2A and p-DAPK expression were performed. The lower panel shows the intensity during basal conditions from the scanned imaged (upper panel). (B) RSC96 cells incubated with and without GAS6 (100 ng/ml) for 30 min and analysed by immunofluorescence. (C) Immunohistochemistry images demonstrated that CIP2A expression levels were higher than sham levels at 14 days and 28 days in the cavernous nerve after BCNI.

Article Snippet: Primary antibodies against GAS6 (1:100, TA323562; Origene), p-AXL (Y779) (1:100, AF2228, R&D), and CIP2A (1:50, NB100-74663, Novus) were used.

Techniques: Incubation, Western Blot, Expressing, Immunofluorescence, Immunohistochemistry

Journal: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury.

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: Figure 4: GAS6 triggers p-Axl, p-DAPK and CIP2A to form a protein complex in RSC96 cells. (A-B) Co-immunoprecipitation between p-Axl, p-DAPK and CIP2A with GAS6 stimulation for 30 min in RSC96 cells. Total lysate indicates 1/10 input in each experiment. The relative quantification of protein expression was normalized with respect to β-actin expression. (C) Immunofluorescence image demonstrating co-localized CIP2A and p-Axl and co-localized p-Axl and p-DAPK (D) after GAS6 stimulation for 30 min in RSC96 cells.

Article Snippet: Primary antibodies against GAS6 (1:100, TA323562; Origene), p-AXL (Y779) (1:100, AF2228, R&D), and CIP2A (1:50, NB100-74663, Novus) were used.

Techniques: Immunoprecipitation, Quantitative Proteomics, Expressing, Immunofluorescence

Journal: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury.

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: Figure 5: GAS6 triggered Schwann cell proliferation primarily through CIP2A and DAPK. (A) RSC96 cells were transfected with either control or DAPK siRNA for 48 h and then exposed to GAS6 (100 ng/ml) for 30 min. Immunoblotting evaluations of pAxl, Axl, CIP2A, DAPK, pERK1/2, ERK1/2, pAKT, AKT, Myc and Survivin. (B) RSC96 cells were transfected with either control or CIP2A siRNA for 48 h and then exposed to GAS6 (100 ng/ml) for 30 min. Immunoblotting evaluations of pAxl, Axl, DAPK, pDAPK, pERK1/2, ERK1/2, pAKT, AKT, Myc and Survivin. (C) DAPK or CIP2A was knocked down for 48 h then RSC96 cells were incubated with GAS6 (100 ng/ml) at the indicated hours. Cell viability was analysed via the WST-1 assay. Data are the mean ± SD, and n = 3 for each time point. * p < 0.05, ** p < 0.01 vs. scramble.

Article Snippet: Primary antibodies against GAS6 (1:100, TA323562; Origene), p-AXL (Y779) (1:100, AF2228, R&D), and CIP2A (1:50, NB100-74663, Novus) were used.

Techniques: Transfection, Control, Western Blot, Incubation, WST-1 Assay

Journal: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury.

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: Figure 6: CIP2A is an inhibitor of PP2A-mediated DAPK dephosphorylation. RSC96 cells were transfected with control, CIP2A siRNA (A), DKK-CIP2A (B), or DAPK siRNA (C) for 48 h and then exposed to GAS6 (100 ng/ml) for 30 min. PP2A activity and immunoblotting evaluations of DKK, CIP2A, pDAPK, and DAPK were performed. (D) RSC96 cells were incubated with GAS6 (100 ng/ ml) for 30 min and co-immunoprecipitation between CIP2A, pDAPK and PP2A was evaluated. Immunoprecipitated PP2A activity was measured. Data are the mean ± SD, and n = 3 for each time point. * p < 0.05, ** p < 0.01, vs. control.

Article Snippet: Primary antibodies against GAS6 (1:100, TA323562; Origene), p-AXL (Y779) (1:100, AF2228, R&D), and CIP2A (1:50, NB100-74663, Novus) were used.

Techniques: De-Phosphorylation Assay, Transfection, Control, Activity Assay, Western Blot, Incubation, Immunoprecipitation

Journal: Oncotarget

Article Title: DAPK and CIP2A are involved in GAS6/AXL-mediated Schwann cell proliferation in a rat model of bilateral cavernous nerve injury.

doi: 10.18632/oncotarget.23978

Figure Lengend Snippet: Figure 7: Schematic diagram of GAS6-induced Schwann cell proliferation. GAS6 binds to its receptor, AXL, and p-AXL induce Schwann cell proliferation via MYC and Survivin signalling. GAS6 increases the expression level of CIP2A to form a p-AXL, p-DAPK and CIP2A protein complex. The presence of CIP2A inhibits the enzyme activity of PP2A and activates downstream ERK1/2 and AKT signals for proliferation.

Article Snippet: Primary antibodies against GAS6 (1:100, TA323562; Origene), p-AXL (Y779) (1:100, AF2228, R&D), and CIP2A (1:50, NB100-74663, Novus) were used.

Techniques: Expressing, Activity Assay

Journal: Mediators of inflammation

Article Title: Growth Arrest-Specific 6 Enhances the Suppressive Function of CD4 + CD25 + Regulatory T Cells Mainly through Axl Receptor.

doi: 10.1155/2017/6848430

Figure Lengend Snippet: Figure 6: Gas6 modulates the Foxp3 and CTLA4 expression mainly through Axl receptor. (a–d) CD4+CD25+Tregs were treated with anti- Axl or anti-Mertk Abs or PBS in the presence of 100 ng/ml rmGas6. After 24 h of incubation, the expression of CTLA-4 and Foxp3 was determined by flow cytometry (𝑛= 4/group). ∗𝑃< 0.05 compared with the value for the Gas6 group, and #𝑃< 0.05 compared with the value for rmGas6+anti-Mertk group. (e–h) The expression of CTLA-4 and Foxp3 in Tregs with or without Axl knockout was determined by flow cytometry. ∗𝑃< 0.05 compared with the value for the Gas6 group.

Article Snippet: To investigate the effect of Gas6 on CD4+CD25+Tregs in vivo, healthy mice were administered 1, 3, or 6 μg/mouse of rmGas6 (8310-GS, R&D Systems, Minneapolis, MN) via tail vein.

Techniques: Expressing, Incubation, Flow Cytometry, Knock-Out

Journal: Molecular Cancer Therapeutics

Article Title: Evaluation of the Role of AXL in Fusion-positive Pediatric Rhabdomyosarcoma Identifies the Small-molecule Inhibitor Bemcentinib (BGB324) as Potent Chemosensitizer

doi: 10.1158/1535-7163.mct-23-0285

Figure Lengend Snippet: Figure 2. Gas6 stimulates Axl signaling in FP-RMS cells. A, Representative Western blot images showing AXL expression in IC-pPDX-104 and Rh41 WT, KO (sgAXL-1) and OE lines. GAPDH was used as loading control. B, Morphologic appearance of IC-pPDX-104 and Rh41 cells with AXL WT, KO and OE. Scale bar, 70 mm. C, Schematic representation of the AXL signaling pathway. D, Representative Western blot images of AXL WT, KO (sgAXL-1) and OE cells stimulated with 400 ng/mL GAS6 for 10 minutes. E, Quantification of Western blots measuring the phosphorylation of downstream targets of AXL in AXL WT, KO (sgAXL-1) and OE cells stimulated with 400 ng/mL GAS6 for 10 minutes. Data are represented as mean SEM of the indicated number of independent biological replicates; ordinary two-way ANOVA with Sidak multiple comparisons test. ns, nonsignificant.

Article Snippet: The next day, we stimulated cells with the indicated concentration of GAS6 (R&D Systems, 885-GSB-050) for 10 minutes at 37 C, and processed them for Western blot analysis.

Techniques: Western Blot, Expressing, Control, Phospho-proteomics

Journal: Molecular Cancer Therapeutics

Article Title: Evaluation of the Role of AXL in Fusion-positive Pediatric Rhabdomyosarcoma Identifies the Small-molecule Inhibitor Bemcentinib (BGB324) as Potent Chemosensitizer

doi: 10.1158/1535-7163.mct-23-0285

Figure Lengend Snippet: Figure 5. Axl contributes to the migratory phenotype of Rh41 cells. A–C, Effect of AXL expression levels on the migratory phenotype of Rh41 cells. Representative images of Rh41 AXL WT (sgAAVS1), KO (sgAXL-1, sgAXL-2) and OE cells (A) and quantification (B) of wound-healing assay. Scale bar, 100 mm. C, Wound area remaining after 20 hours. D–F, Effect of bemcentinib on the migratory phenotype of Rh41 cells. Representative images (D) and quantification (E) of wound-healing assay performed on Rh41 WT cells treated with the indicated concentrations of bemcentinib. Scale bar, 100 mm. F, Wound area remaining after 12 hours in Rh41 cells. G and H, Effect of bemcentinib and AXL stimulation with GAS6 on the migratory phenotype of AXL KO cells. G, Quantification of wound-healing assay performed on Rh41 AXL WT (sgAAVS1) and KO (sgAXL-1, sgAXL-2) cells treated with the indicated concentrations of bemcentinib or with 400 ng/mL GAS6. H, Wound area remaining after 15 hours in Rh41 cells. Data are represented as mean þ SEM of n ¼ 3 biological replicates. Ordinary one-wayANOVA with Dunnett multiple comparison test againstthe control group (WT or 0 mmol/L bemcentinib).

Article Snippet: The next day, we stimulated cells with the indicated concentration of GAS6 (R&D Systems, 885-GSB-050) for 10 minutes at 37 C, and processed them for Western blot analysis.

Techniques: Expressing, Wound Healing Assay, Comparison, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Growth arrest-specific protein 6 (Gas6) pretreatment inhibits transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) in lung epithelial cells (ECs). ( A – C ) LA-4 and ATII ECs were pretreated with 400 ng/mL Gas6 for 20 h prior to 10 ng/mL TGF-β1 treatment for 48 or 72 h. ( A ) Morphological changes in LA-4 ECs were examined by phase-contrast microscopy. Scale bars = 50 μm. Results are representative of three independent experiments. ( B ) Immunoblots of total cell lysates were performed with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies. Densitometry of the relative abundances of the indicated EMT markers. Alpha-tubulin was used as a control. ( C ) The amount of EMT markers’ mRNAs in cell lysates was analysed by real-time PCR and normalized to that of hypoxanthine phosphoribosyltransferase. Values represent the mean ± S.E. of three independent experiments. * P < 0.05; compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

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

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Growth arrest-specific protein 6 (Gas6) pretreatment reduces epithelial-mesenchymal transition (EMT)-regulating transcription factor expression and blocks Smad-independent transforming growth factor (TGF)-β1 signalling in epithelial cells. ( A – C ) LA-4 and ATII epithelial cells (ECs) were pretreated with 400 ng/mL Gas6 20 h prior to 10 ng/mL TGF-β1 stimulation for 48 or 72 h. ( A , B ) The amounts of Snai1/2, Zeb1/2, and Twist1 mRNA were analysed by real-time PCR and normalized to that of hypoxanthine phosphoribosyltransferase ( Hprt ). ( C , D ) Representative immunoblots of LA-4 EC lysates were performed with anti-Snail1, -Zeb1, -total/phosphorylated ERK1/2, and -Akt protein antibodies. Beta-actin or alpha-tubulin was used as a loading control. Densitometric analysis of the indicated protein abundances. Data in all bar graphs are the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

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

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Cyclooxygenase (COX)-2 signaling is required for growth arrest-specific protein 6 (Gas6)-induced production of prostaglandin (PG)E 2 , PGD 2 , and their receptors. ( A – C ) LA-4 and primary alveolar type II (AT II) epithelial cells (ECs) were treated with 400 ng/mL Gas6 for the times indicated. ( A ) qPCR analysis of Cox2 and Cox1 mRNAs in cell lysates. ( B ) Representative immunoblots of LA-4 EC lysates were performed with anti-COX-2, -COX-1, or -α-tubulin antibodies. ( C ) PGE 2 or PGD 2 levels in conditioned media from LA-4 and AT II ECs were measured by enzyme immunoassay. ( D ) Immunoblots of total cell lysates were performed with anti-COX-2 antibodies in LA-4 ECs transfected with COX-2 specific or control siRNA for 6 h. Densitometric analysis of the COX-2 relative abundances. PGE 2 and PGD 2 levels in conditioned media from LA-4 ECs transfected with COX-2 specific or control siRNA for 6 h prior to treatment with 400 ng/mL Gas6 for 20 h were measured by EIA. ( E ) qPCR analysis of Ptger2, Ptger4, Dp1, and Dp2 mRNA in LA-4 ECs treated with 400 ng/mL Gas6 for the time indicated. ( F ) Immunoblot analysis of EP2, EP4, DP1, or DP2 in LA-4 cells. Densitometric analysis of the indicated receptor’ relative abundances. ( G ) qPCR analysis of Ptger2, Ptger4, Dp1, and Dp2 mRNA in LA-4 ECs transfected with COX-2 specific or control siRNA for 6 h prior to treatment with 400 ng/mL Gas6 for 20 h. ( H ) qPCR analysis of Ptger2, Ptger4, Dp1, and Dp2 mRNA in ATII ECs treated with 400 ng/mL Gas6 for the time indicated. Data in all bar graphs are the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Cyclooxygenase (COX)-2-derived signaling mediates growth arrest-specific protein 6 (Gas6)-induced epithelial-mesenchymal transition (EMT) inhibition. ( A – D ) LA-4 ECs were pretreated with 10 μM NS-398 1 h before 400 ng/mL Gas6 treatment for 20 h and then stimulated with 10 ng/mL TGF-β1 treatment for 72 h. ( A ) Morphological changes in the cells were examined by phase-contrast microscopy. Scale bars = 50 μm. ( B ) Immunoblots of total cell lysates were performed with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies. Densitometric analysis of the indicated EMT markers’ relative abundances. ( C , D ) Primary AT II cells were pretreated with 10 μM NS-398 1 h before 400 ng/mL Gas6 treatment for 20 h and then stimulated with 10 ng/mL TGF-β1 treatment for 72 h. qPCR analysis of the mRNAs of EMT markers and EMT-regulating transcription factors. ( E , F ) LA-4 ECs were transfected with COX-2-specific or control siRNAs for 6 h prior to treatment with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 72 h or the times indicated. Representative immunoblots of LA-4 EC lysates were performed with anti-E-cadherin, -N-cadherin, -α-SMA, -total/phosphorylated ERK1/2, and -Akt protein antibodies. Densitometric analysis of the indicated protein abundances. Data in all bar graphs are the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Inhibition, Microscopy, Western Blot, Transfection, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Prostaglandin (PG)E 2 and PGD 2 secretion inhibits growth arrest-specific protein 6 (Gas6)-induced epithelial-mesenchymal transition (EMT) via their receptors. ( A – D ) LA-4 and AT II epithelial cells (ECs) were stimulated with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 48 or 72 h with or without antagonists of EP2 (AH-6809), EP4 (AH-23848), DP1 (BW-A868C), or DP2 (BAY-u3405), each at a dose of 10 μM. ( A ) Representative immunoblots of total cell lysates were performed with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies. ( B ) qPCR analysis of the mRNAs of EMT transcription factors in LA-4 ECs. ( C , D ) qPCR analysis of the mRNAs of EMT markers and EMT transcription factors in primary AT II ECs. Values represent the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Activation of Axl or Mer mediates growth arrest-specific protein 6 (Gas6)-induced inhibition of COX-2 signaling and epithelial-mesenchymal transition (EMT) in LA-4 epithelial cells (ECs). ( A , B ) Immunoblot of total cell lysates were performed with anti-total/phosphorylated Axl and -Mer antibodies in LA-4 ECs treated with 400 ng/mL Gas6 for the times indicated. Densitometric analysis of the indicated protein abundances. ( C , D ) Immunoblots of total cell lysates were performed with anti-Axl, or -Mer antibodies in LA-4 ECs transfected with Axl, Mer, or control siRNA. Densitometric analysis of the indicated protein abundances. ( E – G ) LA-4 ECs were transfected with Axl, Mer, or control siRNAs for 48 h and then stimulated with 400 ng/mL Gas6. ( E , G ) qPCR analysis of the mRNAs of Cox2, Ptger2, Ptger4, Dp1, and Dp2 in LA-4 EC lysates 1 or 20 h after Gas6 stimulation. ( F ) PGE 2 and PGD 2 levels in conditioned media 20 h after Gas6 stimulation were measured by enzyme immunoassay. ( H – J ) LA-4 ECs were transfected with Axl, Mer, or control siRNAs for 48 h prior to treatment with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 72 h or the times indicated. ( H ) Representative immunoblots of total cell lysates with anti-E-cadherin, -N-cadherin, or -α-SMA antibodies in the indicated samples. ( I ) qPCR analysis of the mRNAs of EMT transcription factors. ( J ) Representative immunoblots of total cell lysates with anti-total/phosphorylated ERK1/2 and -Akt protein antibodies. Values represent the mean ± S.E. of three independent experiments. * P < 0.05; compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Inhibition, Western Blot, Transfection, Control, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Gas6 Prevents Epithelial-Mesenchymal Transition in Alveolar Epithelial Cells via Production of PGE 2 , PGD 2 and Their Receptors

doi: 10.3390/cells8070643

Figure Lengend Snippet: Growth arrest-specific protein 6 (Gas6)/Axl signaling inhibits migration and invasion of LA-4 and alveolar type II (AT II) epithelial cells (ECs) via prostaglandin (PG)E 2 and PGD 2 . ( A – D ) LA-4 and primary AT II ECs were stimulated with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 with or without antagonists of EP2 (AH-6809), EP4 (AH-23848), DP1 (BW-A868C), or DP2 (BAY-u3405), each at a dose of 10 μM, for 48 or 72 h. The quantification of migrated or invaded cells in Boyden chambers. ( E , F ) LA-4 ECs were transfected with Axl, Mer, or control siRNAs for 48 h prior to treatment with 400 ng/mL Gas6 for 20 h and then stimulated with 10 ng/mL TGF-β1 for 72 h. The cells were visualized by phase-contrast microscopy for the analysis of migratory in ( E ) left and invasive in ( F ) left abilities using Fn-coated Transwell and Matrigel-coated Transwell plates, respectively. Scale bars: 100 μm. Quantification of cells that migrated in ( E ) right or invaded in ( F ) right. Values represent the mean ± S.E. of three independent experiments. * P < 0.05 compared with control; + P < 0.05 as indicated.

Article Snippet: Recombinant mouse Gas6 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Migration, Transfection, Control, Microscopy