Journal: International journal of molecular sciences

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

doi: 10.3390/ijms25126630

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

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

Techniques: Expressing, Western Blot, Gene Expression

Journal: International journal of molecular sciences

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

doi: 10.3390/ijms25126630

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

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

Techniques: Expressing, Binding Assay, Control, Blocking Assay

Journal: International journal of molecular sciences

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

doi: 10.3390/ijms25126630

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

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

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

Journal: Journal of cellular biochemistry

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

doi: 10.1002/jcb.25582

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

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

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

Journal: Journal of cellular biochemistry

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

doi: 10.1002/jcb.25582

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

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

Techniques: Imaging, Microscopy, Cell Culture, Expressing

Journal: Journal of cellular biochemistry

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

doi: 10.1002/jcb.25582

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

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

Techniques: Expressing, Marker, Western Blot

Journal: Journal of cellular biochemistry

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

doi: 10.1002/jcb.25582

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

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

Techniques: Staining, TUNEL Assay, Western Blot

Journal: Journal of cellular biochemistry

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

doi: 10.1002/jcb.25582

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

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

Techniques: Inhibition, Protein-Protein interactions

Journal: 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 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: 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: Theranostics

Article Title: Regulatory T-cells regulate neonatal heart regeneration by potentiating cardiomyocyte proliferation in a paracrine manner

doi: 10.7150/thno.32734

Figure Lengend Snippet: The most significantly upregulated genes expressed by heart Treg during neonatal heart regeneration. Foxp3 + Treg are purified from the spleen or heart at day 7 post CI to P3 ICR mice. C1: upregulated genes in splenic naïve Treg; C2: upregulated genes in Treg following activation by neoantigens released during cryoinfarction in the heart.

Article Snippet: After that, they were cocultured with in vitro stimulated Treg in a ratio of cardiomyocytes: Treg as 3:1, Treg supernatant-containing dark medium (1:1) or 50 ng/ml murine CCL24 (Biolegend, 585102), 100 ng/ml murine GAS6 (RnD systems, 8310-GS-050), 1 ug/ml murine GRN (Lifespan biosciences, LS-G3786-10) or 100 ng/ml murine amphiregulin (RnD systems, 989-AR-100) at 37°C for 1 day before analysis.

Techniques: Purification, Activation Assay, Immunopeptidomics

Journal: Theranostics

Article Title: Regulatory T-cells regulate neonatal heart regeneration by potentiating cardiomyocyte proliferation in a paracrine manner

doi: 10.7150/thno.32734

Figure Lengend Snippet: Treg directly promote proliferation of mouse neonatal cardiomyocytes in a paracrine manner. Immunocytochemistry for cTnT + (red) and Ki67 + (green), pH3 + (green) or Aurora B + (green) cells at day 1 after coculture of (A) CD3 + CD4 + hCD2 + Treg, Treg supernatant (SN), or (G) the combination of CCL24, GAS6 and AREG (Pool 3) with mouse neonatal cardiomyocytes of P1 ICR hearts, scale bars: 50 um. Quantification of (B) the absolute number of total cTnT + cardiomyocytes after cocultured for 3 days; or (C) %Ki67 + cTnT + , (D) %pH3 + cTnT + or (E) %Aurora B + cTnT + proliferating cardiomyocytes among total cTnT + cardiomyocytes based on (A). Quantification of proliferating cardiomyocytes after cultured with (F) the respective paracrine factors or (H-J) Pool 3 for 1 day. Data are presented as mean±S.D., n = 3 independent experiments, *P<0.05, **P<0.01.

Article Snippet: After that, they were cocultured with in vitro stimulated Treg in a ratio of cardiomyocytes: Treg as 3:1, Treg supernatant-containing dark medium (1:1) or 50 ng/ml murine CCL24 (Biolegend, 585102), 100 ng/ml murine GAS6 (RnD systems, 8310-GS-050), 1 ug/ml murine GRN (Lifespan biosciences, LS-G3786-10) or 100 ng/ml murine amphiregulin (RnD systems, 989-AR-100) at 37°C for 1 day before analysis.

Techniques: Immunocytochemistry, 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: 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

Journal: PLoS ONE

Article Title: Gas6/Axl signaling attenuates alveolar inflammation in ischemia-reperfusion-induced acute lung injury by up-regulating SOCS3-mediated pathway

doi: 10.1371/journal.pone.0219788

Figure Lengend Snippet: (A) Lung weight gain, (B) pulmonary microvascular permeability (Kf), (C) ratios of lung wet/dry (W/D) weight, (D) lung weight/body weight (LW/BW), and (E) protein concentration in bronchoalveolar lavage fluid (BALF) in IR-ALI. The increase of these parameters in the IR group was significantly attenuated by pretreatment with Gas6. IR: ischemia–reperfusion. Data are expressed as the mean ± SD (n = 6 per group). * P < 0.05 compared with the control group; ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; # P < 0.05 compared with the IR group; ## P < 0.01 compared with the IR group; ### P < 0.001 compared with the IR group.

Article Snippet: After 24 h of incubation, the cells were pretreated with vehicle and 5, 10, or 20 ng/ml of mouse recombinant Gas6 protein (986-GS/CF, R&D Systems, Minneapolis, MN, USA).

Techniques: Permeability, Protein Concentration, Control

Journal: PLoS ONE

Article Title: Gas6/Axl signaling attenuates alveolar inflammation in ischemia-reperfusion-induced acute lung injury by up-regulating SOCS3-mediated pathway

doi: 10.1371/journal.pone.0219788

Figure Lengend Snippet: (A) CINC-1, (B) IL-1β, (C) IL-6, and (D) TNF-α in IR-ALI. Gas6 attenuated the production of proinflammatory cytokines induced by IR. IR: ischemia–reperfusion. Data are expressed as the means ± SD (n = 6 per group). * P < 0.05 compared with the control group; ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; # P < 0.05 compared with the IR group; ### P < 0.001 compared with the IR group.

Article Snippet: After 24 h of incubation, the cells were pretreated with vehicle and 5, 10, or 20 ng/ml of mouse recombinant Gas6 protein (986-GS/CF, R&D Systems, Minneapolis, MN, USA).

Techniques: Control

Journal: PLoS ONE

Article Title: Gas6/Axl signaling attenuates alveolar inflammation in ischemia-reperfusion-induced acute lung injury by up-regulating SOCS3-mediated pathway

doi: 10.1371/journal.pone.0219788

Figure Lengend Snippet: (A) Hematoxylin and eosin staining for lung tissue (200× magnification), (B) neutrophil count, and (C) lung injury score in IR-ALI. Gas6 attenuates the severity of IR-ALI. IR: ischemia–reperfusion. Data are expressed as the mean ± SD (n = 6 per group). ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; ### P < 0.001 compared with the IR group.

Article Snippet: After 24 h of incubation, the cells were pretreated with vehicle and 5, 10, or 20 ng/ml of mouse recombinant Gas6 protein (986-GS/CF, R&D Systems, Minneapolis, MN, USA).

Techniques: Staining, Control

Journal: PLoS ONE

Article Title: Gas6/Axl signaling attenuates alveolar inflammation in ischemia-reperfusion-induced acute lung injury by up-regulating SOCS3-mediated pathway

doi: 10.1371/journal.pone.0219788

Figure Lengend Snippet: (A) Bronchoalveolar lavage fluid (BALF) TNF-α, (B) BALF IL-6, (C) NF-κB p65 levels of lung tissues, and (D) cytoplasmic IκB-α levels of lung tissues in IR-ALI. Gas6 modulates TNF-α and IL-6 levels in BALF and NF-κB expressions of lung tissues in IR-ALI. IR: ischemia–reperfusion. Data are expressed as the means ± SD (n = 6 per group for BALF and N = 4 per group for lung tissue). ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; # P < 0.05 compared with the IR group; ## P < 0.01 compared with the IR group; ### P < 0.001 compared with the IR group.

Article Snippet: After 24 h of incubation, the cells were pretreated with vehicle and 5, 10, or 20 ng/ml of mouse recombinant Gas6 protein (986-GS/CF, R&D Systems, Minneapolis, MN, USA).

Techniques: Control

Journal: PLoS ONE

Article Title: Gas6/Axl signaling attenuates alveolar inflammation in ischemia-reperfusion-induced acute lung injury by up-regulating SOCS3-mediated pathway

doi: 10.1371/journal.pone.0219788

Figure Lengend Snippet: (A) SOCS3 protein levels in lung tissues determined by western blot analysis. (B) SOCS3 mRNA expressions in lung tissues. (C) Representative images of SOCS3 immunofluorescence staining (FITC-labeled green; original magnification ×400) of rat lung. Nuclei were counterstained with DAPI (blue). SOCS3 expressions of alveolar epithelium are significantly decreased after IR and restored by Gas6 treatment. IR: ischemia–reperfusion. Data are expressed as the mean ± SD (n = 4 per group). ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; ## P < 0.01 compared with the IR group; ### P < 0.001 compared with the IR group.

Article Snippet: After 24 h of incubation, the cells were pretreated with vehicle and 5, 10, or 20 ng/ml of mouse recombinant Gas6 protein (986-GS/CF, R&D Systems, Minneapolis, MN, USA).

Techniques: Western Blot, Immunofluorescence, Staining, Labeling, Control

Journal: PLoS ONE

Article Title: Gas6/Axl signaling attenuates alveolar inflammation in ischemia-reperfusion-induced acute lung injury by up-regulating SOCS3-mediated pathway

doi: 10.1371/journal.pone.0219788

Figure Lengend Snippet: (A-B) SOCS3, TRAF6, cytoplasmic IκB-α, and nuclear NF-κB p65 levels determined by western blot analysis. TATA and β-actin served as loading controls for nuclear and cytoplasmic proteins, respectively. Gas6 up-regulates SOCS3 and down-regulates TRAF6 and NF-κB in HR model. HR: hypoxia-reoxygenation. Data are expressed as the means ± SD (n = 4 per group). * P < 0.05 compared with the control group; ** P < 0.01 compared with the control group; # P < 0.05 compared with the HR group; ## P < 0.01 compared with the HR group; ### P < 0.001 compared with the HR group.

Article Snippet: After 24 h of incubation, the cells were pretreated with vehicle and 5, 10, or 20 ng/ml of mouse recombinant Gas6 protein (986-GS/CF, R&D Systems, Minneapolis, MN, USA).

Techniques: Western Blot, Control

Journal: PLoS ONE

Article Title: Gas6/Axl signaling attenuates alveolar inflammation in ischemia-reperfusion-induced acute lung injury by up-regulating SOCS3-mediated pathway

doi: 10.1371/journal.pone.0219788

Figure Lengend Snippet: (A) T-Axl protein levels determined by western blot analysis. (B) Axl mRNA expressions. (C) p-Axl, (D) SOCS3 and (E) nuclear NF-κB p65 levels determined by western blot analysis. HR resulted in lower p-Axl and SOCS3 levels and a higher NF-κB p65 level. Pretreatment with the Axl inhibitor R428 reversed the effects of Gas6 on p-Axl, SOCS3 and NF-κB p65. Data are expressed as the means ± SD (n = 4 per group). T-Axl: total Axl. p-Axl: phosphorylated Axl. HR: hypoxia-reoxygenation. Gas6: recombinant Gas6 20 ng/ml. R428: R428 100 nM. All values are expressed as the means ± SD (n = 4 per group). * P < 0.05 compared with the control group; *** P < 0.001 compared with the control group; ## P < 0.01 compared with the HR group.

Article Snippet: After 24 h of incubation, the cells were pretreated with vehicle and 5, 10, or 20 ng/ml of mouse recombinant Gas6 protein (986-GS/CF, R&D Systems, Minneapolis, MN, USA).

Techniques: Western Blot, Recombinant, Control

Journal: PLoS ONE

Article Title: Gas6/Axl signaling attenuates alveolar inflammation in ischemia-reperfusion-induced acute lung injury by up-regulating SOCS3-mediated pathway

doi: 10.1371/journal.pone.0219788

Figure Lengend Snippet: (A) Gas6 mRNA expressions in lung tissues. (B) Endogenous Gas6 levels in perfusate determined by ELISA analysis. (C) p-Axl levels determined by western blot analysis. Recombinant Gas6, but not endogenous Gas6, up-regulated phosphorylation of Axl in IR-ALI. p-Axl: phosphorylated Axl. IR: ischemia–reperfusion. Data are expressed as the mean ± SD (n = 4 per group for tissue mRNA and n = 6 per group for perfusate). * P < 0.05 compared with the control group; ** P < 0.01 compared with the control group; *** P < 0.001 compared with the control group; # P < 0.05 compared with the IR group; ### P < 0.001 compared with the IR group.

Article Snippet: After 24 h of incubation, the cells were pretreated with vehicle and 5, 10, or 20 ng/ml of mouse recombinant Gas6 protein (986-GS/CF, R&D Systems, Minneapolis, MN, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant, Phospho-proteomics, Control