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: 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: 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

Journal: Journal of Neuroinflammation

Article Title: Gas6 induces inflammation and reduces plaque burden but worsens behavior in a sex-dependent manner in the APP/PS1 model of Alzheimer’s disease

doi: 10.1186/s12974-022-02397-y

Figure Lengend Snippet: Effect of Gas6 overexpression on open field task and novel object recognition task. A Schematic of AAV-Gas6 construct containing full length Gas6 protein and AAV-control which contains an attenuated Gas6 sequence without the Gla and EGF domains. B Schematic of experimental design: APP/PS1 male and female mice received bilateral hippocampal injections of either AAV-Gas6 or AAV-control at 9 months of age. Behavioral tests were run 1 month later, and all mice were sacrificed at 10 months of age. C Mice were allowed to freely explore a 31 × 31 cm box for 5 min, during which distance travelled and time spent in the center of the box were quantified. N = 7–10 mice per group. Error bars represent mean ± SEM. Two-way ANOVA, # p = 0.0594, * p = 0.0225, ** p = 0.0026. D Mice performed a novel object recognition task in which they were allowed to freely explore a box with two identical objects. Two hours later, they were exposed to one familiar object and one novel object, and time spent exploring each object was measured. Discrimination index: ((Time spent with novel object − Time spent with familiar object)/(Total time spent with both objects)). N = 7–10 mice per group. Error bars represent mean ± SEM

Article Snippet: Gas6(Myc-DDK-tagged)-mouse growth arrest specific 6 (Gas6) cDNA was purchased from OriGene (Rockville, MD) and used to construct an AAV-packaging vector using In-Fusion HD Cloning Kit (Clontech Laboratories).

Techniques: Over Expression, Construct, Control, Sequencing

Journal: Journal of Neuroinflammation

Article Title: Gas6 induces inflammation and reduces plaque burden but worsens behavior in a sex-dependent manner in the APP/PS1 model of Alzheimer’s disease

doi: 10.1186/s12974-022-02397-y

Figure Lengend Snippet: Gas6 overexpression worsens contextual fear conditioning performance in male APP/PS1 mice. A Schematic of contextual and cued fear conditioning task. B , C Freezing responses in each 30 s interval for male and female mice. N = 7–10 mice per group. Shaded areas represent mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons, *indicates APP-Gas6 group significantly differs from APP-control group for indicated 30 s time frame ( p < 0.05). D , E Freezing responses in each 30 s interval for male and female mice during cued fear conditioning task in novel context. Vertical line indicates onset of conditioned tone. N = 7–10 mice per group. Upper and lower borders of shaded areas represent mean ± SEM

Article Snippet: Gas6(Myc-DDK-tagged)-mouse growth arrest specific 6 (Gas6) cDNA was purchased from OriGene (Rockville, MD) and used to construct an AAV-packaging vector using In-Fusion HD Cloning Kit (Clontech Laboratories).

Techniques: Over Expression, Control

Journal: Journal of Neuroinflammation

Article Title: Gas6 induces inflammation and reduces plaque burden but worsens behavior in a sex-dependent manner in the APP/PS1 model of Alzheimer’s disease

doi: 10.1186/s12974-022-02397-y

Figure Lengend Snippet: Gas6 expression reduces plaque number in male APP/PS1 mice. A Representative images from male and female APP mice injected with AAV-Gas6 or AAV-control at 9 months of age and injected with MX-04 24 h before sacrifice at 10 mo. Scale bar is 100 μm. B , C Quantitative measurements of % area of MX-04 and plaque numbers. N = 7–10 mice per group. Error bars represent mean ± SEM. Unpaired two-tailed t -test ** p = 0.0014. D Particle analysis on MX-04 positive plaques. N = 7–10 mice per group. Error bars represent mean ± SEM. Two-way ANOVA ** p < 0.01

Article Snippet: Gas6(Myc-DDK-tagged)-mouse growth arrest specific 6 (Gas6) cDNA was purchased from OriGene (Rockville, MD) and used to construct an AAV-packaging vector using In-Fusion HD Cloning Kit (Clontech Laboratories).

Techniques: Expressing, Injection, Control, Two Tailed Test, Particle Size Analysis

Journal: Journal of Neuroinflammation

Article Title: Gas6 induces inflammation and reduces plaque burden but worsens behavior in a sex-dependent manner in the APP/PS1 model of Alzheimer’s disease

doi: 10.1186/s12974-022-02397-y

Figure Lengend Snippet: Effect of Gas6 on CD68 and Iba-1 expression in APP/PS1 mice. A Representative images and quantification of CD68 ( B ) and Iba-1 ( C ) staining on tissue from AAV-Gas6 or AAV-control mice. Scale bar represents 20 μm. N = 7–11 per group, 5–6 plaques per animal. Error bars represent mean ± SEM. Unpaired two-tailed t -test # p = 0.0693

Article Snippet: Gas6(Myc-DDK-tagged)-mouse growth arrest specific 6 (Gas6) cDNA was purchased from OriGene (Rockville, MD) and used to construct an AAV-packaging vector using In-Fusion HD Cloning Kit (Clontech Laboratories).

Techniques: Expressing, Staining, Control, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Gas6 induces inflammation and reduces plaque burden but worsens behavior in a sex-dependent manner in the APP/PS1 model of Alzheimer’s disease

doi: 10.1186/s12974-022-02397-y

Figure Lengend Snippet: Gas6 does not affect microglial phagocytosis of amyloid beta in vivo or in vitro. A Ten-month-old APP/PS1 and nontransgenic littermates injected with AAV-Gas6 or AAV-control at 9 months of age were intraperitoneally injected with MX-04 24 h prior to sacrifice. Brains were processed and microglia (CD45 lo /CD11b+) were analyzed by flow cytometry. Representative plots of gating strategy. B, C MX-04 + microglial populations showing percent of MX-04+ cells and relative median fluorescence per cell. N = 2–4 per group. Error bars represent mean ± SEM. D Primary microglia from Axl +/− mice were treated with rmGas6 (50 nM) and beta-amyloid (1–42) HiLyte Fluor 488 for 15 or 30 min and analyzed by flow cytometry. N = 4–5 biological replicates per group. Error bars represent mean ± SEM. E Primary microglia from Axl + / − or Axl−/− P0-P2 pups were treated with rmGas6 for 15, 30, 60, or 120 min and analyzed by flow cytometry. N = 4–6 biological replicates per group. Error bars represent mean ± SEM

Article Snippet: Gas6(Myc-DDK-tagged)-mouse growth arrest specific 6 (Gas6) cDNA was purchased from OriGene (Rockville, MD) and used to construct an AAV-packaging vector using In-Fusion HD Cloning Kit (Clontech Laboratories).

Techniques: In Vivo, In Vitro, Injection, Control, Flow Cytometry, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Gas6 induces inflammation and reduces plaque burden but worsens behavior in a sex-dependent manner in the APP/PS1 model of Alzheimer’s disease

doi: 10.1186/s12974-022-02397-y

Figure Lengend Snippet: RNAseq of AAV-Gas6 treated APP/PS1 mice microglia reveals an enrichment of transcriptional signatures related to interferon response. A PCA plot of microglial samples. B Heatmap showing significantly DEGs between microglia isolated from AAV-Gas6 treated APP/PS1 mice and AAV-control hippocampi. Colors are shown in logarithmic scale. C Volcano plot with significantly downregulated genes (shown in red) and significantly upregulated genes (shown in green). Genes with a p -value < 0.001 are labeled. Vertical line is the p = 0.05 cutoff and horizontal lines show log2FoldChange = − 1 or 1. D Overrepresentation test of significantly upregulated genes through clusterProfiler package shows enrichment for interferon-gamma and beta response related pathways. E , F CXCL13 and CCL2 ELISA on hippocampal protein lysates from Gas6 and control-treated mice. Two-way ANOVA followed by Tukey post-hoc ** p = 0.0018, # p = 0.0878

Article Snippet: Gas6(Myc-DDK-tagged)-mouse growth arrest specific 6 (Gas6) cDNA was purchased from OriGene (Rockville, MD) and used to construct an AAV-packaging vector using In-Fusion HD Cloning Kit (Clontech Laboratories).

Techniques: Isolation, Control, Labeling, Enzyme-linked Immunosorbent Assay

Journal: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 2. Regulation of Axl shedding. L929 (A) and L929R (B) cells were treated with 200 ng/ml of PMA for different times, and the concentration of sAxl in cell-conditioned medium was evaluated by a specific ELISA. Untreated cells were used as controls. *, P 0.05 versus control samples. L929 (C) and L929R (D) cells were left untreated or treated with PMA for 2 h. Expression of membrane-bound Axl was evaluated by flow cytometry. (E and F) Cells were treated with PMA (200 ng/ml), Gas6 (50 ng/ml), IL-15 (50 ng/ml), TNF- (10 ng/ml), and LPS (10 ng/ml) for 2 h, and the concentration of sAxl in the culture medium was quantified by ELISA. Untreated (medium) cells were used as controls. **, P 0.01 versus control samples.

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Control, Expressing, Membrane, Cytometry

Journal: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 7. sAxl is present in mouse serum and associates with Gas6. (A) Sera from C57BL/6, CH3, BALB/c, Axl/, Axl/, and Axl/

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 8. Immobilized Axl-Fc chimeric protein promotes cell migra- tion and induces the phosphorylation of Axl and PI3K. (A) L929R cells were plated onto Axl-Fc- or IL-3R–Fc (control)-coated six-well plates and allowed to grow in the presence of 10% FCS. After 18 h, a wound was created by scratching with a pipette tip (0 h). The cells were washed with PBS and incubated further to allow migration into the wounded area. Phase-contrast images of matched pairs of marked wound regions were taken 6 and 18 h later to assess cell migration. (B) L929R cells were serum starved for 4 h and incubated in Axl-Fc- coated wells for 15 min. Incubation in IL-3R–Fc (control)-coated wells or stimulation with 100 ng/ml of Gas6 was used as a negative or positive control, respectively. Cells were lysed, and Axl or PI3K was immunoprecipitated from the lysates with specific Abs. Precipitates were subjected to 10% SDS-PAGE and analyzed with anti-pTyr Abs. Detection of Axl or PI3K on the same blots was used as a loading control. The picture is representative of three independent experi- ments, all of which yielded highly comparable results. IP, immunopre- cipitation; WB, Western blotting.

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques: Phospho-proteomics, Control, Transferring, Incubation, Migration, Positive Control, Immunoprecipitation, SDS Page, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum

doi: 10.1128/mcb.25.21.9324-9339.2005

Figure Lengend Snippet: FIG. 9. Immobilized Axl-Fc chimeric protein induces cell migration and phosphorylation of Axl and PI3K in WT but not Axl/ MEFs. (A) MEFs were plated onto Axl-Fc- or IL-3R–Fc (control)-coated six-well plates. A confluent cell monolayer was wounded by scratching with a pipette tip (0 h). The cells were washed with PBS and incubated further to allow migration into the wounded area. Phase-contrast images of matched pairs of marked wound regions were taken 6 and 18 h later to assess cell migration. (B) MEFs were serum starved for 4 h and incubated in Axl-Fc-coated wells for 15 min. Incubation in IL-3R–Fc-coated (control) wells or stimulation with 100 ng/ml of Gas6 was used as a negative or positive control, respectively. Cells were lysed, and Axl or PI3K was immunoprecipitated from the lysates with specific Abs. Precipitates were subjected to 10% SDS-PAGE and analyzed with anti-pTyr Abs. Detection of Axl or PI3K on the same blots was used as a loading control. IP, immunoprecipitation; WB, Western blotting.

Article Snippet: Recombinant murine Gas6, rat antimouse Gas6, biotinylated goat anti-mouse Gas6, Axl, Mer, and Tyro3 (Dtk) Abs and Axl-Fc and IL-3R–Fc chimeras were purchased from R&D Systems.

Techniques: Migration, Phospho-proteomics, Control, Transferring, Incubation, Positive Control, Immunoprecipitation, SDS Page, Western Blot

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