Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 1. Differentiation of THP-1 cells induced by PMA. (A) THP-1 monocytes are differentiated to macrophages and induced to express Mertk by the addition of 100 ng/mL of PMA for at least 24 h, while Axl expression is not induced. Likewise, Tyro3 expression remains stable and is unaffected by treatment. H1299 and Beas2B cells are used as positive controls for Mertk/Axl and Tyro3, respec- tively. The observed doublet for Mertk corresponds to fully glycosylated and partially glycosylated glycoforms. (B) THP-1 cells treated with 100 ng/mL of PMA over 48 h exhibit a decreased expression of Gas6 but an increased expression of Protein S. (C) MertK and Gas6 expression patterns over a time of 48 h after 100 ng/mL of PMA treatment as quantified from Western blots. (D) Bar plots showing MerTK and Gas6 expression quantified from Western blots at 0 h and post 72 h of 100 ng/mL of PMA treatment (significant differences were observed between the 0 h and 72 h time points for Gas6 (* p = 0.029) and MerTK (** p = 0.003), as determined by independent t-tests). (E) THP-1 cells were treated with 100 ng/mL of PMA over 72 h. mRNA was analyzed via qRT-PCR for Mertk, Axl, Protein S (ProS), Tyro3, and Gas6. Mertk transcription was highly elevated, more than 10-fold, due to PMA treatment, while Gas6 transcription was repressed more than 10-fold. (F) PMA treatment of THP-1s illustrates a complimentary phenomenon. As a monocyte, Gas6 is expressed with little Mertk expression; however, when differentiated, Mertk is highly expressed in favor of Gas6.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 3. γ-carboxylated Gas6 Reduces Full-length Mertk Expression and Decreases sMertk. (A) γ- carboxylation status (Gla) of Gas6 is regulated with the addition of Vitamin K or warfarin, producing either a γ-carboxylated, active ligand or a non-γ-carboxylated, inactive ligand, respectively. The carboxylation status (Gla) domain of Gas6 can bind with externalized PS. (B) Recombinant inactive (Gas6-W) and active (Gas6-VK) were produced via HEK293 transfection, with a mock transfection control (Mock). The amount of Gas6 was observed (bottom), while the carboxylation status that is responsible for ligand activity was determined for each (left, top). (C) PMA-differentiated THP-1s were treated for 4 h with either serum-free RPMI only (-), a mock transfection control, 10 nM of active Gas6 (Gas6-VK), or 10 nM of inactive Gas6 (Gas6-W). Treatments were alone or combined with inhibitors of 3 µM of GW280264X (an ADAM17 inhibitor), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a proteasomal inhibitor). (D) Quantitative results indicate that Gas6, either active or inactive, does not stabilize the C-terminal fragments as shown in the DAPT and MG132 treatments (bands at 75 kDa). As denoted by sMertk (top), the cleavage is decreased with GW280264X treatment compared to the untreated control.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Expressing, Recombinant, Produced, Transfection, Control, Activity Assay

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 5. γ-carboxylated Gas6 reduces the tagged Mertk construct on cell membranes of THP-1 cells. (A) THP-1s expressing the tagged construct were starved for 18 h in serum-free RPMI and treated for 3 h. Flow cytometry data shows positive GFPs without staining. As expected, CHX treatment after 3 h reduces the expression of the construct. Gas6-VK, used at a concentration > 10 nM, decreases the FLAG-PE signal more when compared to other treatments known to induce cleavage (PMA, LPS). GW280264X is used as a control for Mertk cleavage. (B) Histogram analysis of “A.” Gas6-VK induces a shift in the FLAG-PE signal, showing that less surface Mertk is present. γ-Carboxylated Gas6 induced the degradation of Mertk on the cell membrane. (C) THP-1s treated with γ-Carboxylated Gas6 at a 10 nM concentration show increased MerTK phosphorylation, detected by immunoblotting against pMerTK. (D) THP-1s expressing the tagged construct, treated with >10 nM of Gas6-VK + 1 µM of PS, show a reduced level of both domains of the tagged construct, suggesting that the Gas6-VK + PS treatment is leading to a degradation of the full-length receptor.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Construct, Expressing, Flow Cytometry, Staining, Concentration Assay, Control, Membrane, Phospho-proteomics, Western Blot

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 6. Confocal imaging of the GFP-tagged MerTK construct displayed the differential localization of MerTK upon ligand stimulation and the inhibition of proteases. (A) DAPT and MG132 treatments induce increased cytoplasmic GFP signals compared to untreated cells. (B) Confocal imaging shows GFP localized on the cell membrane, indicating the presence of tagged Mertk constructs on the cell surface. γ-Carboxylated Gas6-treated tagged THP-1 cells showed a reduction in the MerTK construct from the membrane and the localization in lysosomes. (C) Quantification of the GFP fluorescence intensity per cell, calculated from confocal images in mock, γ-carboxylated Gas6, and non-γ-carboxylated Gas6, with the bar plots showing the mean and standard error of each treatment. ns; non-significant; *** p < 0.001; **** p < 0.0001.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Imaging, Construct, Inhibition, Membrane, Fluorescence

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 7. γ-carboxylation of Gas6 induces Mertk degradation independent of phosphorylation. (A) Mutants of the tagged construct were created. K619M, a substitution in the ATP-binding site (underlined) of the Mertk kinase domain, inhibits autophosphorylation by preventing ATP binding and the exchange of phosphate molecules. * Amino acid position for ADAM17 cleavage. (B) Constructs are treated with 10 nM ofGas6-VK for 30 min after a 6 h serum starvation. With the ability to bind ATP and phosphorylate, the WT construct becomes phosphorylated while the kinase dead K619M mutant does not. (C) Mutant constructs were treated with 3 µM of GW280264X (an ADAM17 inhibitor; GW), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a protea- somal inhibitor) for 4 h. Results show that the absence of the K619M C-terminal fragment from the MG132 treatment with the addition of GW indicates the fragment is a product of cleavage. (D) Confocal images indicated that the K619M mutants have more cytoplasmatic c-Mertk than WT Mertk with MG132 treatment. (E) Comparison of the contrasting pathways between Notch Recep- tor and MerTK Signaling Models. In the absence of a ligand, Notch is not cleaved, while MerTK undergoes homeostatic cleavage, leading to proteasomal degradation. Upon ligand binding, Notch is cleaved at the ADAM 17 site, revealing the gamma-secretase site. Subsequent gamma-secretase cleavage releases the Notch intracellular domain, translocating it to the nucleus for transcriptional activation. Conversely, MerTK, upon ligand (Gas6) interaction, is internalized into endosomal compartments and localizes within lysosomes.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Phospho-proteomics, Construct, Binding Assay, Mutagenesis, Comparison, Ligand Binding Assay, Activation Assay

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: 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: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Gas6 delays senescence in vascular smooth muscle cells through the PI3K/ Akt/FoxO signaling pathway.

doi: 10.1159/000373940

Figure Lengend Snippet: Fig. 2. Gas6 delays the senescence process in VSMCs. (A and B) Western blots demonstrating that cells trea- ȋʹͷͲȀȌ͵ϐ ͳ Ͷ and p21Cip1 expression. (C) The ef- fects of Gas6 on the IS and RS models were examined using western blotting. In the IS model, the Gas6-tre- ated cells showed low p21Cip1 and p16 Ͷ expression. In the RS model, the Gas6-treated cells also showed low p21Cip1 and p16 ͶǤȋȌǦȾǦ Ǧ ϐ Ǧ Ǧ RS models. (E and F) When these cells were treated with Axl-Fc, the levels of p16 Ͷ and p21Cip1 and the ǦȾǦ ϐ Ǥ (n=3 in each case). The values are presented as the mean±SD. *P<0.05, **P<0.01 and ***P<0.001 compared with the corresponding blank group; #P<0.05, ##P<0.01 and ###P<0.001 compared with the corresponding blank group. Bar, 200 μm.

Article Snippet: Recombinant mouse Gas6 (Gas6) protein and the recombinant mouse Axl-Fc protein fragment (Axl-Fc) were purchased from R&D Systems (St. Paul, MN, USA).

Techniques: Western Blot, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Gas6 delays senescence in vascular smooth muscle cells through the PI3K/ Akt/FoxO signaling pathway.

doi: 10.1159/000373940

Figure Lengend Snippet: Fig. 3. Axl is the primary receptor in the Gas6-mediated anti-senescence effect. (A) Western blotting and SA- ȾǦͳ Ͷ and p21Cip1ϐ in R428-treated cells regardless of Gas6 treatment compared with the two R428-free cell groups, whereas ϐ ͶʹͺǦ ǤȋȌǦȾǦ ͶʹͺǦ ϐ compared with the two R428-free cell groups. All the results shown are from representative experiments (n=3 in each case). The values are presented as the mean±SD. **P<0.01 and ***P<0.001 compared with the non-R428-treated group; ##P<0.01 and ###P<0.001 compared with the non-R428-treated group. Bar, 200 μm.

Article Snippet: Recombinant mouse Gas6 (Gas6) protein and the recombinant mouse Axl-Fc protein fragment (Axl-Fc) were purchased from R&D Systems (St. Paul, MN, USA).

Techniques: Western Blot

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Gas6 delays senescence in vascular smooth muscle cells through the PI3K/ Akt/FoxO signaling pathway.

doi: 10.1159/000373940

Figure Lengend Snippet: Fig. 4. Gas6 promotes the transition from G1 to S phase. Cell cycle analyses showing that Gas6-treated cells in both the IS and the RS models showed higher percentages of S phase and a lower percentage of G1 phase Ǧ Ǧ Ǣȋ Ȍϐ with the corresponding controls. (B) EdU staining results showed that the positive staining rates in both the IS and the RS models after Gas6 treatment were higher than in controls. All the results shown are from representative experiments (n=3 in each case). The values are presented as the mean±SD. *P<0.05 compared with the non-Gas6-treated group; #P<0.05 compared with the non-Gas6-treated group.

Article Snippet: Recombinant mouse Gas6 (Gas6) protein and the recombinant mouse Axl-Fc protein fragment (Axl-Fc) were purchased from R&D Systems (St. Paul, MN, USA).

Techniques: Staining

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

Journal: PloS one

Article Title: Dual involvement of growth arrest-specific gene 6 in the early phase of human IgA nephropathy.

doi: 10.1371/journal.pone.0066759

Figure Lengend Snippet: Figure 1. Gas6 expression in human IgA nephropathy. Biopsy samples were immunostained using indirect immunohistochemistry procedure with anti-Gas6 antibody. Representative images are shown in (A) from control patients, (B) from IgA nephropathy cases immunostained in epithelial cells, and (C) from IgA nephropathy cases immunostained in endothelial and mesangial cells. The original magnification was X200. Endo/mes, Endothelial and mesangial. doi:10.1371/journal.pone.0066759.g001

Article Snippet: Three times diluted samples were then applied on the ELIZA plate (human Gas6 Duoset ELIZA Development kit, R&D Systems).

Techniques: Expressing, Immunohistochemistry, Control

Journal: PloS one

Article Title: Dual involvement of growth arrest-specific gene 6 in the early phase of human IgA nephropathy.

doi: 10.1371/journal.pone.0066759

Figure Lengend Snippet: Figure 2. Gas6 stained area in human IgA nephropathy. Gas6 stained area is expressed as a percentage of total glomerular area occupied by Gas6 immunostained area. For each sample, at least eight glomerular profiles per patient were measured. IgA epi, IgA epithelial type. doi:10.1371/journal.pone.0066759.g002

Article Snippet: Three times diluted samples were then applied on the ELIZA plate (human Gas6 Duoset ELIZA Development kit, R&D Systems).

Techniques: Staining

Journal: PloS one

Article Title: Dual involvement of growth arrest-specific gene 6 in the early phase of human IgA nephropathy.

doi: 10.1371/journal.pone.0066759

Figure Lengend Snippet: Figure 3. Correlation of Gas6 stained area with prognostic factors. Gas6 stained area in human IgA nephropathy correlated with (A) Oxford mesangial hypercellularity score and (B) urine protein excretion. N = 28. Oxford M score, Oxford mesangial hypercellularity score. doi:10.1371/journal.pone.0066759.g003

Article Snippet: Three times diluted samples were then applied on the ELIZA plate (human Gas6 Duoset ELIZA Development kit, R&D Systems).

Techniques: Staining

Journal: PloS one

Article Title: Dual involvement of growth arrest-specific gene 6 in the early phase of human IgA nephropathy.

doi: 10.1371/journal.pone.0066759

Figure Lengend Snippet: Figure 4. Inverse correlation of Gas6 stained area with p27 positive cell number. (A) Biopsy samples were immunostained using indirect immunohistochemistry procedure with anti-p27 antibody. p27 positive cell number was counted. The result was divided by glomeruli number. For each sample, at least eight glomerular profiles per patient were measured. p27 positive cell number correlated with Oxford mesangial hypercellularity score. (B) Gas6 stained area inversely correlated with p27 positive cell number. N = 28. Oxford M score, Oxford mesangial hypercellularity score. doi:10.1371/journal.pone.0066759.g004

Article Snippet: Three times diluted samples were then applied on the ELIZA plate (human Gas6 Duoset ELIZA Development kit, R&D Systems).

Techniques: Staining, Immunohistochemistry

Journal: PloS one

Article Title: Dual involvement of growth arrest-specific gene 6 in the early phase of human IgA nephropathy.

doi: 10.1371/journal.pone.0066759

Figure Lengend Snippet: Figure 6. Double immunohistochemistry of Dtk and Axl with cell specific markers or Gas6. Biopsy samples were immunostained using indirect immunohistochemistry procedure with (A) anti-Dtk, anti-Nephrin (a podocyte marker), (B) anti-Dtk, anti-Gas6 or (C) anti-Axl, anti-CD34 (an endothelial cell marker), (D) anti-Axl, anti-Gas6 antibody. (A,B) Dtk immunostaining was detected in both control and IgA nephropathy, mostly merged with Nephrin. It was also merged with Gas6 in epithelial type IgA nephropathy. (C,D) Axl immunostaining was detected in both control and IgA nephropathy, mostly merged with CD34. It was also merged with Gas6 in endothelial and mesangial type IgA nephropathy. X200. IgAN, IgA nephropathy. IgA epi, IgA epithelial type. IgA endo/mes, IgA endothelial and mesangial type. doi:10.1371/journal.pone.0066759.g006

Article Snippet: Three times diluted samples were then applied on the ELIZA plate (human Gas6 Duoset ELIZA Development kit, R&D Systems).

Techniques: Immunohistochemistry, Marker, Immunostaining, Control

Journal: PloS one

Article Title: Dual involvement of growth arrest-specific gene 6 in the early phase of human IgA nephropathy.

doi: 10.1371/journal.pone.0066759

Figure Lengend Snippet: Figure 7. Effect of Gas6 on Dtk and p27 expression in podocytes. Podocytes were differentiated and stimulated with recombinant Gas6 or TGFb1 for 24 hours. Cell lysates were subjected to immunoblotting with the antibodies indicated. Then, podocytes expressed Dtk. Gas6 could increase the expression of Dtk and reduce p27, as well as TGFb1. Representative data were shown from four independent experiments. Quantitative examinations of the band density for (B) Dtk and (C) p27 were shown. The columns and error bars are the mean 6 SD of four independent experiments. doi:10.1371/journal.pone.0066759.g007

Article Snippet: Three times diluted samples were then applied on the ELIZA plate (human Gas6 Duoset ELIZA Development kit, R&D Systems).

Techniques: Expressing, Recombinant, Western Blot

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 cancer research : MCR

Article Title: Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow

doi: 10.1158/1541-7786.MCR-17-0132

Figure Lengend Snippet: (A–C) PCa cells (PC3 and DU145) cultured with NE, Propranolol, or vehicle control and evaluated for Ki67 expression using flow cytometry (N=3; Student’s t-test). (D–E) Immunocytochemistry for Ki67 expression by PCa cells following in vitro culture of PC3 cells and GAS6+/+ OB co-culture in the presence of NE or vehicle control (N=3; Student’s t-test). (F–G) PC3-FUCCI cells cultured in vitro with NE or vehicle control and evaluated for cell cycle phase using flow cytometry (N=3; Student’s t-test).

Article Snippet: ELISA An ELISA assay was performed following the manufacturer’s instructions (R&D Systems, Dy986) to evaluate GAS6 expression in OBs (MC3T3-E1s and primary murine OBs) in the presence of vehicle or NE.

Techniques: Cell Culture, Control, Expressing, Flow Cytometry, Immunocytochemistry, In Vitro, Co-Culture Assay

Journal: Molecular cancer research : MCR

Article Title: Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow

doi: 10.1158/1541-7786.MCR-17-0132

Figure Lengend Snippet: (A) Expression of mRNA dormancy-associated genes and adhesion/homing-associated genes expressed by OBs were determined in MC3T3-E1 OB cultures treated with NE or vehicle control using qPCR (N=2; Student’s t-test). Values were normalized to GAPDH levels. (C) Expression of GAS6 in PCa cells (PC3) and OBs (MG63, MC3T3-E1, and GAS6+/+ OB) was determined by an ELISA in culture conditions with NE and vehicle control (N=3; student’s t-test). (D) Ki67 expression of OBs (MG63, MC3T3-E1, and GAS6+/+ OBs) cultured with NE or vehicle control was determined by flow cytometry (N=3; Student’s t-test).

Article Snippet: ELISA An ELISA assay was performed following the manufacturer’s instructions (R&D Systems, Dy986) to evaluate GAS6 expression in OBs (MC3T3-E1s and primary murine OBs) in the presence of vehicle or NE.

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

Journal: Molecular cancer research : MCR

Article Title: Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow

doi: 10.1158/1541-7786.MCR-17-0132

Figure Lengend Snippet: (A–B) GAS6 expression in GAS6+/+ OBs cultured under conditions with NE or vehicle control was determined by immunocytochemistry and quantified (N=3; student’s t-test). GAS6−/− OBs were cultured under the same conditions, serving as a negative control. (C) PC3 cells and (D) DU145 cells were co-cultured with GAS6+/+ and GAS6−/− OBs in the presence of NE or vehicle control. Ki67 expression was determined by FACS (N=3; ANOVA). (E) PC3-FUCCI cells were co-cultured with GAS6+/+ and GAS6−/− OBs in the presence of NE or vehicle control. Cell cycle phase was determined by FUCCI vector expression using flow cytometry (N=3; ANOVA).

Article Snippet: ELISA An ELISA assay was performed following the manufacturer’s instructions (R&D Systems, Dy986) to evaluate GAS6 expression in OBs (MC3T3-E1s and primary murine OBs) in the presence of vehicle or NE.

Techniques: Expressing, Cell Culture, Control, Immunocytochemistry, Negative Control, Plasmid Preparation, Flow Cytometry

Journal: Molecular cancer research : MCR

Article Title: Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow

doi: 10.1158/1541-7786.MCR-17-0132

Figure Lengend Snippet: (A) Pictorial diagram demonstrating the experimental protocol for the ex vivo culture model. (B,C) Representative sample of the flow plot gates found from PC3-FUCCI cells injected into ex vivo GAS6+/+ femurs treated with vehicle (B) or NE (C) added to the culture conditions. Live cells were negatively gated for murine IgG2b b haplotype (mH-2Db) (BioLegend, Cat# 111516, PE/Cy7) and mCD45 (BioLegend, Cat# 103112, APC), which were then positively gated for HLA-A,B,C (Biolegend, Cat# 311426, APC/Cy7). After these gates were applied, cells were plotted on the FUCCI spectrum. (D) PC3-FUCCI cells were isolated following injection into ex vivo GAS6+/+ or GAS6 −/− femur explants and cell cycle was determined using flow cytometry (N=3; Student’s t-test).

Article Snippet: ELISA An ELISA assay was performed following the manufacturer’s instructions (R&D Systems, Dy986) to evaluate GAS6 expression in OBs (MC3T3-E1s and primary murine OBs) in the presence of vehicle or NE.

Techniques: Ex Vivo, Injection, Isolation, Flow Cytometry

Journal: Molecular cancer research : MCR

Article Title: Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow

doi: 10.1158/1541-7786.MCR-17-0132

Figure Lengend Snippet: Model of NE signaling within the bone marrow may activate dormant PCa DTCs through both direct and indirect methods. The direct method hypothesis includes NE binding to PCa DTCs via Adrenergic Receptors, inhibiting cell cycle inhibitors p21, p27 and p38 causing PCa entry into the proliferative cell cycle. The indirect method hypothesis includes NE binding to local OBs via Adrenergic Receptors, resulting in decreased secretion of the dormancy regulating molecule GAS6. ADR = Adrenergic Receptors; NE= Norepinephrine; PCa =Prostate Cancer; DTC = Disseminated Tumor Cell; OBs = Osteoblast Cell.

Article Snippet: ELISA An ELISA assay was performed following the manufacturer’s instructions (R&D Systems, Dy986) to evaluate GAS6 expression in OBs (MC3T3-E1s and primary murine OBs) in the presence of vehicle or NE.

Techniques: Binding Assay