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: 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: Aging (Albany NY)

Article Title: GAS6 ameliorates advanced age-associated meiotic defects in mouse oocytes by modulating mitochondrial function

doi: 10.18632/aging.203328

Figure Lengend Snippet: Reduced expression of Gas6 and increased meiotic errors in oocytes with maternal aging. ( A , B ) Typical expression patterns of the Gas6 transcripts ( A ) and protein ( B ) in GV and MII oocytes from young and aged female mice, respectively. Gas6 expression was significantly decreased in aged oocytes. The levels of the GAS6 protein are presented in a bar graph. The data are presented as the means ± SEM. Different letters indicate significant differences at p <0.05. Young oocytes were obtained from 3-week-old female mice; aged oocytes were obtained from 12-month-old female mice. ( C ) Maternal aging causes spindle organization defects and chromosome misalignment during oocyte maturation. Green, spindle; blue, chromosome; red, mitochondria; red box, oocyte with abnormal spindle and chromosome alignment. The scale bars represent 25 μm.

Article Snippet: Recombinant mouse GAS6 protein was purchased from R&D Systems (8310-GS; Minneapolis, MN, USA).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: GAS6 ameliorates advanced age-associated meiotic defects in mouse oocytes by modulating mitochondrial function

doi: 10.18632/aging.203328

Figure Lengend Snippet: GAS6 ameliorates age-associated meiotic defects in oocytes. ( A ) Micrographs of aged MII oocytes treated without or with the GAS6 protein. Control, nontreated MII oocyte; GAS6-rescued, MII oocyte treated with the GAS6 protein. The scale bar represents 100 μm. ( B ) In vitro maturation rates of aged mouse oocytes after the microinjection of the GAS6 protein into GV-stage oocytes. After GAS6 restoration, these oocytes developed into morphologically normal MII oocytes, similar to the control groups. The data are presented as the means ± SEM. ( C , D ) Microinjection of the GAS6 protein led to a partial recovery of the decreased GAS6 levels induced by maternal aging. α-TUBULIN was used as a loading control. Relative expression levels of the GAS6 protein are presented in a bar graph and compared to the control young oocyte group. The data are presented as the means ± SEM. Different letters indicate significant differences at p <0.05. Control, nontreated oocyte; GAS6-rescued, oocyte treated with the GAS6 protein; Young, oocytes obtained from 3-week-old female mice; Aged, oocytes obtained from 12-month-old female mice. ( E ) Immunofluorescence staining for spindles and chromosomes in aged MII oocytes after injection of the GV oocytes without (control) or with the GAS6 protein (GAS6-rescued), which were then allowed to mature in vitro . Green, spindle; blue, chromosome; red, mitochondria; red box, oocyte with abnormal spindle and chromosome alignment. The scale bars represent 25 μm. ( F ) Proportions of control or GAS6 protein-injected MII oocytes with defects in spindle assembly and chromosome configuration.

Article Snippet: Recombinant mouse GAS6 protein was purchased from R&D Systems (8310-GS; Minneapolis, MN, USA).

Techniques: Control, In Vitro, Microinjection, Expressing, Immunofluorescence, Staining, Injection

Journal: Aging (Albany NY)

Article Title: GAS6 ameliorates advanced age-associated meiotic defects in mouse oocytes by modulating mitochondrial function

doi: 10.18632/aging.203328

Figure Lengend Snippet: Improvements in mitochondrial function in oocytes during aging after GAS6 supplementation. ( A ) Measurements of the mtDNA copy numbers in aged oocytes expressing GAS6. The mtDNA copy number was higher in GAS6-rescued aged oocytes than in control aged oocytes. Different letters indicate significant differences at p <0.05. Control, nontreated oocyte; GAS6-rescued, oocyte treated with the GAS6 protein; Young, oocytes obtained from 3-week-old female mice; Aged, oocytes obtained from 12-month-old female mice. ( B ) Expression of Mtnd1 and Mtatp6 in aged MII oocytes from the control and GAS6-rescued groups. Different letters indicate significant differences at p <0.05. ( C ) Representative images of ΔΨm in aged MII oocytes after GAS6 supplementation. ΔΨm indicates the ratio of RITC (J-aggregate, high membrane potential) to FITC (J-monomer, low membrane potential) intensity in GAS6-rescued aged MII oocytes. Scale bars represent 50 μm. ( D ) Graphic representation of the results shown in C. The data are presented as the means ± SEM. Different letters indicate significant differences at p <0.001. ( E ) Effects of GAS6 restoration on ATP levels in aging oocytes. Increasing GAS6 expression resulted in elevated mitochondrial ATP production. Different letters indicate significant differences at p <0.05. ( F ) Treatment with GAS6 reduced ROS levels in young and aged MII oocytes. After treatment, MII oocytes were cultured in M16 medium supplemented with DCFH-DA to evaluate ROS levels. Scale bars represent 100 μm. ( G ) GAS6 treatment increased oocyte GSH levels with aging. The data are presented as the means ± SEM. Different letters indicate significant differences at p <0.05. ( H ) The ratio of GSH to GSSG in GAS6-rescued aged MII oocytes was calculated. The GSH/GSSG ratio is a biochemical marker of oxidative stress. The GSH/GSSG ratio was increased in aged MII oocytes after the restoration of GAS6. Thus, GAS6 reduces the oxidative stress caused by maternal aging. Different letters indicate significant differences at p <0.05.

Article Snippet: Recombinant mouse GAS6 protein was purchased from R&D Systems (8310-GS; Minneapolis, MN, USA).

Techniques: Expressing, Control, Membrane, Cell Culture, Marker

Journal: Aging (Albany NY)

Article Title: GAS6 ameliorates advanced age-associated meiotic defects in mouse oocytes by modulating mitochondrial function

doi: 10.18632/aging.203328

Figure Lengend Snippet: GAS6 restores the Gas6 silencing- and/or maternal age-induced decline in fertility. ( A ) Microphotographs of PN embryos after in vitro fertilization. Aged oocytes were treated without (control) or with the GAS6 protein (GAS6-rescued) and then fertilized after in vitro culture. White triangles indicate PN formation. Scale bars represent 100 μm. ( B ) Percentage of oocytes showing PN formation after in vitro fertilization. Experiments were repeated at least three times, and data are presented as the means ± SEM. Different letters indicate significant differences at p <0.05.

Article Snippet: Recombinant mouse GAS6 protein was purchased from R&D Systems (8310-GS; Minneapolis, MN, USA).

Techniques: In Vitro, Control

Journal: Aging (Albany NY)

Article Title: GAS6 ameliorates advanced age-associated meiotic defects in mouse oocytes by modulating mitochondrial function

doi: 10.18632/aging.203328

Figure Lengend Snippet: GAS6 attenuates the decrease in the quality of aged oocytes. Aged oocytes showed increased mitochondrial deterioration, elevated oxidative stress and insufficient cytoplasmic maturation due to decreased Gas6 expression, resulting in meiotic defects and consequential PN formation failure. Moreover, GAS6-rescued aged oocytes collectively exhibited metabolic alterations, indicating better-quality mitochondrial functions. Therefore, the age-associated decreases in oocyte quality and fertility were prevented by restoring GAS6 levels.

Article Snippet: Recombinant mouse GAS6 protein was purchased from R&D Systems (8310-GS; Minneapolis, MN, USA).

Techniques: Expressing

Journal: EMBO Reports

Article Title: The actin modulator hMENA regulates GAS 6‐ AXL axis and pro‐tumor cancer/stromal cell cooperation

doi: 10.15252/embr.202050078

Figure Lengend Snippet: A Heat map of proteins identified by LC‐MS/MS analysis as differentially secreted in CM of normal fibroblasts (P‐NF#1 and P‐NF#2), CAFs with low level of hMENAΔv6 expression (P‐CAFs low#44 and #49), CAFs with high level of hMENAΔv6 expression (P‐CAFs high #67 and #36).The boxed hMENAΔv6 associated signature represents proteins exclusively present in the CM derived from CAFs high . Color code is shown in the upper left corner. Co‐variance = 0.2, q ‐value = 0.1 (10% false discovery rate), adjusted P value = 0.0074542. N = 36. The values of mass spec identified for each protein were plotted in log 2 scale. B Quantification of GAS6 secretion levels, as detected by ELISA, in the CM of P‐NF (#1), P‐CAF low (#44 and #49) and P‐CAF high (#67 and #36). Data are presented as the mean ± SD of two biological replicates. Statistical analysis was performed with one‐way ANOVA P < 0.0001, followed by Bonferroni's multiple comparison test. *** P < 0.001. C Real‐time qRT–PCR analysis of the relative GAS6 mRNA expression level in NFs (P‐NF#1), P‐CAF low and P‐CAF high , as described above. Data are presented as the mean ± SD of three replicates. Statistical analysis was performed with one‐way ANOVA P = 0.001, followed by Bonferroni's multiple comparison test. * P < 0.05, ** P < 0.01. D Real‐time qRT–PCR analysis of the relative GAS6 mRNA expression level in PANC‐1 and P‐CAF ( n = 3), showing a low GAS6 expression in PANC‐1 cells. Data are presented as the mean ± SD. P values were calculated by two‐sided Student's t ‐test. *** P < 0.001. E Boxplots showing the mRNA expression of GAS6 in normal lung fibroblasts (white) ( n = 15) versus primary NSCLC fibroblasts (light blue) ( n = 15) (GSE22862 data set). In each boxplot the median value (horizontal line), 25 th –75 th percentiles (box outline), and highest and lowest values within 1.5× of the interquartile range (vertical line) are shown. Statistical significance was calculated by Mann–Whitney U ‐test (two‐sided) ( P = 0.0208). F, G Real‐time qRT–PCR analysis of P#138 CAF (F) and L#189 CAF (G) transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)) as representative cases. The siRNA‐mediated knock‐down of hMENA/hMENAΔv6 resulted in a significant reduction of GAS6 mRNA expression levels compared to siCNT cells (set as 100). Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t ‐test ** P < 0.01, *** P < 0.001. H Quantification of Matrigel invasion assay of PANC‐1 cultured for 48 h with DMEM (culture medium), or conditioned medium (CM) derived from control siRNA P#106 CAFs (siCNT P‐CAF-CM), GAS6 siRNA (siGAS6 P-CAF‐CM), hMENA(t) siRNA (sihMENA(t) P-CAF‐CM), hMENA(t) siRNA plus rGAS6 (sihMENA(t) P‐CAF-CM + rGAS6). Number of invaded PANC‐1 cells after 48 h of treatment was measured by counting 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one‐way ANOVA P = 0.004, followed by Bonferroni's multiple comparison test. * P < 0.05, ** P < 0.01.

Article Snippet: Recombinant GAS6 (885‐GS; R&D Systems) was added to cell media of PANC‐1 cells at 200 ng/ml, according to the effect evidenced by a dose curve.

Techniques: Liquid Chromatography with Mass Spectroscopy, Expressing, Derivative Assay, Mass Spectrometry, Enzyme-linked Immunosorbent Assay, Comparison, Quantitative RT-PCR, MANN-WHITNEY, Transfection, Control, Knockdown, Invasion Assay, Cell Culture

Journal: EMBO Reports

Article Title: The actin modulator hMENA regulates GAS 6‐ AXL axis and pro‐tumor cancer/stromal cell cooperation

doi: 10.15252/embr.202050078

Figure Lengend Snippet: Immunoblot of AXL expression in PANC‐1 and A549 cancer cells upon transfection of control siRNA (CNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a reduction of AXL protein expression. Real‐time qRT–PCR analysis of the relative AXL mRNA expression in PANC‐1 and A549 cancer cells transfected with control siRNA (siCNT) or hMENA(t) siRNA (sihMENA(t)), indicating that the knock‐down of hMENA isoforms, resulted in a significant reduction of mRNA AXL expression. Data are presented as the mean ± SD of three replicates. P values were calculated by two‐sided Student's t ‐test. ** P < 0.01, *** P < 0.001. Real‐time qRT–PCR analysis of the relative levels of mature AXL mRNA or AXL pre‐mRNA in PANC‐1 (left) and A549 (right) cell lines, transfected with control siRNA (siCNT) or hMENA(t) siRNA, sihMENA(t). Data represent percent of AXL mRNA or pre‐mRNA levels in hMENA(t) silenced cells relative to siCNT control cells (set at 100). Data are presented as the mean ± SD of two biological replicates. P values were calculated by two‐sided Student's t ‐test. * P < 0.05, *** P < 0.001. Immunoblot analysis with the indicated antibodies of PANC‐1 cells transfected with control siRNA (CNT) or hMENA(t) siRNA, showing that the knock‐down of total hMENA isoforms, (hMENA(t)) inhibits GAS6‐mediated pAXL and pAKT expression. Cells were serum starved overnight and subsequently stimulated with DMSO (0.02%) in control culture medium (−) or rGAS6 (200 ng/ml), for 30 and 60 min. The fold change of pAXL or pAKT expression respect to siCNT untreated cells is reported. Quantification of in vitro Matrigel invasion assay of PANC‐1 siCNT cells (siCNT) and hMENA/hMENAΔv6 silenced cells (sihMENA(t)) toward rGAS6 as chemo‐attractant (siCNT + rGAS6 and sihMENA(t) + rGAS6), showing that the knock‐down of hMENA(t) reduced cancer cell invasion toward GAS6. The number of invading cells was counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one‐way ANOVA P < 0.0001, followed by Bonferroni's multiple comparison test. ** P < 0.01. Quantification of in vitro Matrigel invasion assay of PANC‐1 siCNT cells (siCNT) and hMENA/hMENAΔv6 silenced cells (sihMENA(t)), untreated (−) or treated with conditioned media derived from CAFs (P‐CAF#36‐CM) for 48 h. The number of invaded cells was counted in 6 random fields. Data are presented as the mean ± SD of three biological replicates. Statistical analysis was performed with one‐way ANOVA P < 0.0001, followed by Bonferroni's multiple comparison test. *** P < 0.001. Source data are available online for this figure.

Article Snippet: Recombinant GAS6 (885‐GS; R&D Systems) was added to cell media of PANC‐1 cells at 200 ng/ml, according to the effect evidenced by a dose curve.

Techniques: Western Blot, Expressing, Transfection, Control, Knockdown, Quantitative RT-PCR, In Vitro, Invasion Assay, Comparison, Derivative Assay

Journal: EMBO Reports

Article Title: The actin modulator hMENA regulates GAS 6‐ AXL axis and pro‐tumor cancer/stromal cell cooperation

doi: 10.15252/embr.202050078

Figure Lengend Snippet: Overall survival (OS) curves in pancreatic adenocarcinoma patients (PDAC) ( n = 172) from The Cancer Genome Atlas (TCGA) showed that combined expression of AXL, GAS6, and ENAH (hMENA) is a prognostic signature in PDAC. Overall survival (OS) curves in lung squamous cancer patients (LUSC) ( n = 501) from The Cancer Genome Atlas (TCGA) showed that combined expression of AXL, GAS6, and ENAH (hMENA) is a prognostic signature in LUSC. Data information: In (A, B) patients were stratified in three groups on the basis of the AXL/GAS6/ENAH (left), AXL/GAS6 (middle), and ENAH (right) signature expression levels. P values are shown. Statistical significance was calculated by using the log‐rank test.

Article Snippet: Recombinant GAS6 (885‐GS; R&D Systems) was added to cell media of PANC‐1 cells at 200 ng/ml, according to the effect evidenced by a dose curve.

Techniques: Expressing

Journal: ASN NEURO

Article Title: Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination

doi: 10.1177/1759091416668430

Figure Lengend Snippet: Expression of TAM receptors and Gas6 in different mouse brain regions and at different postnatal ages. (a) qPCR expression analysis of mRNA for Gas6 and TAM receptors. Values represent mean ± SEM of relative gene expression; n = 4 for all samples except for optic nerve ( n = 3) and P14 cerebellum ( n = 2). All samples were normalized against Cdc40 as internal control; * p < .05, ** p < .01, *** p < .001, **** p < .0001 for comparisons as indicated. Columns containing letter a are significant compared with cortex of the same age, and columns containing letter b are significant compared with cerebellum of the same age. (b) Representative Western blot of Axl, Tyro3, and GAPDH (loading control) proteins. Lanes correspond to the following: (1) P7 cortex, (2) P7 cerebellum, (3) P14 cortex, (4) P14 cerebellum, (5) adult cortex, (6) adult cerebellum, and (7) adult optic nerve. The histograms show the quantification of Axl and Tyro3 protein expression by densitometric analysis. Values represent mean ± SEM ( n = 4 blots); * p < .05, # p = .056 between samples as indicated by lines. Columns containing letter a are significant compared with cortex of the same age, and columns containing letter b are significant compared with cerebellum of the same age.

Article Snippet: For experiments, recombinant Gas6 protein (diluted in cell culture medium containing Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine; Lonza, Slough, UK) was added directly to the culture medium at 400 ng/ml final concentration.

Techniques: Expressing, Gene Expression, Control, Western Blot

Journal: ASN NEURO

Article Title: Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination

doi: 10.1177/1759091416668430

Figure Lengend Snippet: Expression of Tyro3, Axl, and Gas6 mRNA in human glial cell cultures. Extracted mRNA from human astrocytes and human oligodendrocyte precursors were analyzed by qPCR using specific primers for human Tyro3, Axl, and Gas6 genes. Results were analyzed based on 2 −ΔCt method.

Article Snippet: For experiments, recombinant Gas6 protein (diluted in cell culture medium containing Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine; Lonza, Slough, UK) was added directly to the culture medium at 400 ng/ml final concentration.

Techniques: Expressing

Journal: ASN NEURO

Article Title: Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination

doi: 10.1177/1759091416668430

Figure Lengend Snippet: Effect of Gas6 on number of OPCs or oligodendrocytes in cultured mouse optic nerves. (a) Confocal images of optic nerves treated with mock medium (−Gas6) and Gas6, showing Sox10+ cells with green fluorescence. (b) Cell counts from images taken from optic nerves treated with Gas6 in the absence or presence of two Gas6 antagonists: soluble Axl receptor (Axl/Fc) and Warfarin-Gas6. Values represent mean ± SEM (control and Gas6, n = 8; Axl/Fc + Gas6, n = 2; Warfarin-Gas6, n = 4; n represents number of separate experiments); *** p < .001 versus control ( SEM values are as follows: Mock = 5.0648, Gas6 = 3.5669, Axl/Fc + Gas6 = 4.0488, Warfarin-Gas6 = 3.4071, analysis of variance with Bonferroni correction).

Article Snippet: For experiments, recombinant Gas6 protein (diluted in cell culture medium containing Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine; Lonza, Slough, UK) was added directly to the culture medium at 400 ng/ml final concentration.

Techniques: Cell Culture, Fluorescence, Control

Journal: ASN NEURO

Article Title: Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination

doi: 10.1177/1759091416668430

Figure Lengend Snippet: Gas6 significantly attenuates demyelination in a toxin-induced demyelination model. (a) Cerebellar slice cultures were treated with vehicle (methanol), LPC + Mock medium (−Gas6), and LPC + Gas6 and stained with MBP antibody. For each treatment within an experiment, one cerebellar slice was used, and one field of view per slice was analyzed. Images were taken from the top end of the lobules were the axons spread out. Scale bar = 50 µm. (b) Quantification of myelination through the number of MBP + axons with lengths greater than 50 µm. Values represent mean ± SEM ( n = 6 experiments); * p < .05 for comparison indicated, analysis of variance with Bonferroni correction.

Article Snippet: For experiments, recombinant Gas6 protein (diluted in cell culture medium containing Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine; Lonza, Slough, UK) was added directly to the culture medium at 400 ng/ml final concentration.

Techniques: Staining, Comparison

Journal: ASN NEURO

Article Title: Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination

doi: 10.1177/1759091416668430

Figure Lengend Snippet: Effect of Gas6 on activation of intracellular STAT3 and myelination through MBP expression in cultured optic nerves. Representative Western blots are shown of proteins (a) pSTAT3 and (b) MBP in optic nerve lysates. The graphs accompanying each blot show the densitometric quantification of protein levels relative to GAPDH protein in those samples. Values represent mean ± SEM ( n = 6 blots); * p < .05, ** p < .01 versus control, Student t test. Gas6 significantly increased the phosphorylation of STAT3 as well as MBP in optic nerve cultures.

Article Snippet: For experiments, recombinant Gas6 protein (diluted in cell culture medium containing Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine; Lonza, Slough, UK) was added directly to the culture medium at 400 ng/ml final concentration.

Techniques: Activation Assay, Expressing, Cell Culture, Western Blot, Control, Phospho-proteomics

Journal: ASN NEURO

Article Title: Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination

doi: 10.1177/1759091416668430

Figure Lengend Snippet: Gas6 activates STAT3 in mature oligodendrocytes and astrocytes and activates Tyro3 receptor after 3 h stimulation in culture. (a) Cerebellar slices were treated with mock or Gas6 medium for 3 h, and fixed sections were double stained for p-STAT3 and APC (mature oligodendrocyte marker), p-STAT3 and GFAP (astrocyte marker), p-STAT3 and NeuN (neuronal marker; scale bar = 20 µm). The stainings reveal that p-STAT3 is present in oligodendrocytes and astrocytes but not in neurons. The arrows point to cells that are both GFAP+ and p-STAT3+. (b) Representative Western blot of p-Tyro3 protein in optic nerve lysate. The graph shows the densitometric quantification of protein level relative to actin in the same sample. Values represent mean ± SEM ( n = 5 blots); ** p < .01 versus control, Student t test. Gas6 significantly increased the activation of Tyro3.

Article Snippet: For experiments, recombinant Gas6 protein (diluted in cell culture medium containing Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine; Lonza, Slough, UK) was added directly to the culture medium at 400 ng/ml final concentration.

Techniques: Staining, Marker, Western Blot, Control, Activation Assay

Journal: ASN NEURO

Article Title: Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination

doi: 10.1177/1759091416668430

Figure Lengend Snippet: Summary of Genes in Cultured Mouse Optic Nerves Altered by Gas6 Stimulation by ≥2-Fold.

Article Snippet: For experiments, recombinant Gas6 protein (diluted in cell culture medium containing Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine; Lonza, Slough, UK) was added directly to the culture medium at 400 ng/ml final concentration.

Techniques: Cell Culture

Journal: ASN NEURO

Article Title: Gas6 Promotes Oligodendrogenesis and Myelination in the Adult Central Nervous System and After Lysolecithin-Induced Demyelination

doi: 10.1177/1759091416668430

Figure Lengend Snippet: Effect of Gas6 on expression of selected MS-related genes in cultured mouse optic nerves. qRT-PCR was performed on extracts from mock and Gas6-treated optic nerve cultures, using specific primer or probe sets, and normalizing expression against the GAPDH gene. Treatment of optic nerves with Gas6 resulted in downregulation of Epha1 , GFAP , and MMP9 genes. Values represent mean ± SEM ( n = 4 for qPCR experiments for all genes except for Epha1 ( n = 2)).

Article Snippet: For experiments, recombinant Gas6 protein (diluted in cell culture medium containing Dulbecco’s Modified Eagle Medium, supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine; Lonza, Slough, UK) was added directly to the culture medium at 400 ng/ml final concentration.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR