Journal: bioRxiv

Article Title: EndoNB: A general strategy to study the internalization of cell surface proteins

doi: 10.1101/2025.06.08.658482

Figure Lengend Snippet: a , Structural alignment of the hybrid and β-I domains of wild-type and Alfa-tagged Integrin β1. b , Schematics showing the effect of 12G10 and Mab13 antibodies on integrin β1 activation state. These antibodies act by stabilizing the integrins in their active or inactive state. c , Quantification of integrin β1 uptake by flow cytometry using EndoNB in the presence of no antibodies (no treatment), or in the presence of 12G10 (activating), Mab13 (inactivating) or unrelated (anti integrin αvβ3 antibody). (n = 12, each n represents the median fluorescence of 2000-8000 cells). d , Structural alignment of the extracellular region of wild-type and Alfa-tagged transferrin receptor (TFR). Wild type: PDB 1suv, while Alfa-tagged was generated by AlphaFold 3. See for comparison of Alfa-tagged dimeric and monomeric TFR. e , Schematics showing the experimental design to test if transferrin receptor uptake is stimulated by the presence of transferrin. f , Quantification of transferrin receptor uptake for 15 or 30 minutes by flow cytometry using EndoNB in the presence of increasing concentrations of transferrin. (n = 4, each n represents the median fluorescence of 2000-8000 cells). See for quantification of transferrin-AlexaFluor488 signals at each condition. g , Structural alignment of the transmembrane and extracellular region of wild-type and Alfa-tagged AXL. Both structures generated with AlphaFold 3. h , Quantification of AXL uptake by flow cytometry using EndoNB in the absence or presence of the AXL ligand GAS6 (1ug/ml). (n = 4, each n represents the median fluorescence of 2000-8000 cells). See for comparison with the conditions with and without 3C. ns = non-significant, * p> 0.05, **** p> 0.0001. ANOVA with Tukey’s post hoc analysis.

Article Snippet: SNAP-JF646 substrate was a kind gift by Luke Lavis via the open chemistry team (jJanelia); Transferrin-Alexa Fluor 488 (Thermo Fisher scientific, #T13342); Phalloidin-iFluor 488 (AAT Bioquest, #23115); GAS6 recombinant protein (MedChemExpress via Nordic Biosite HY-P700724-20).

Techniques: Activation Assay, Flow Cytometry, Fluorescence, Generated, Comparison

Journal: bioRxiv

Article Title: EndoNB: A general strategy to study the internalization of cell surface proteins

doi: 10.1101/2025.06.08.658482

Figure Lengend Snippet: a , Structural alignment of the monomeric and dimeric extracellular region of wild-type and Alfa-tagged transferrin receptor (TFR, PDB). Wild type: PDB 1suv, while Alfa-tagged was generated by AlphaFold 3. b , Quantification of transferrin (AlexaFluor488) in each condition measured in . c , Quantification of AXL uptake by flow cytometry using EndoNB in the absence or presence of the AXL ligand GAS6 (1µg/ml) and also in the presence or not of 3C. (n = 4, each n represents the median fluorescence of 2000-8000 cells). ns = non-significant, ** p> 0.01, *** p> 0.001, **** p> 0.0001. ANOVA with Tukey’s post hoc analysis.

Article Snippet: SNAP-JF646 substrate was a kind gift by Luke Lavis via the open chemistry team (jJanelia); Transferrin-Alexa Fluor 488 (Thermo Fisher scientific, #T13342); Phalloidin-iFluor 488 (AAT Bioquest, #23115); GAS6 recombinant protein (MedChemExpress via Nordic Biosite HY-P700724-20).

Techniques: Generated, Flow Cytometry, Fluorescence

Journal: Cells

Article Title: Corosolic Acid Attenuates the Invasiveness of Glioblastoma Cells by Promoting CHIP-Mediated AXL Degradation and Inhibiting GAS6/AXL/JAK Axis

doi: 10.3390/cells10112919

Figure Lengend Snippet: Involvement of GAS6-associated cascade in CA-inhibited migration and invasion of GBM cells. ( A ) Cells were treated with CA (20 μM) and then lysed for immunodetection of the indicated targets by Western blotting. ( B , C ) Cells were treated with CA (20 μM), GAS6 (100 ng/mL), or a combination of CA and GAS6, and then lysed for immunodetection of the indicated targets ( B ) or F-actin ( C ) by Western blotting. ( D ) Cells were treated with CA (20 μM), GAS6 (100 ng/mL) or a combination of CA and GAS6, then subjected to migration and invasion assay. Chemiluminescence signal was semi-quantitated by densitometric analysis, and GAPDH was used as an internal control. * and ** p < 0.05 and 0.01, respectively, compared with the control (DMSO-treated cells). # p < 0.05 compared with CA alone. Images were acquired using a light microscope at 200× magnification.

Article Snippet: Recombinant Human GAS6 Protein (Rh-GAS6) was purchased from R&D Systems, Inc (Minneapolis, MN, USA)

Techniques: Migration, Immunodetection, Western Blot, Invasion Assay, Control, Light Microscopy

Journal: Cells

Article Title: Corosolic Acid Attenuates the Invasiveness of Glioblastoma Cells by Promoting CHIP-Mediated AXL Degradation and Inhibiting GAS6/AXL/JAK Axis

doi: 10.3390/cells10112919

Figure Lengend Snippet: Molecular docking analysis and proposed mechanism for CA-inhibited invasiveness of GBM cells. ( A ) Superposition of GAS6 (green) and template CA (cyan), hydro-gen bonding interactions with GAS6 and CA. Binding affinity: −7.6 kcal/mol. ( B ) Superposition of AXL (green) and template CA (cyan), hydrogen bonding interactions with AXL and CA. Binding affinity: −5.6 kcal/mol. Interacting amino acid residues: Asn677, Arg676, Leu542 and Asp672. ( C ) Proposed mechanism for CA-inhibited invasiveness of GBM cells.

Article Snippet: Recombinant Human GAS6 Protein (Rh-GAS6) was purchased from R&D Systems, Inc (Minneapolis, MN, USA)

Techniques: Binding 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