recombinant mouse gas6 protein  (R&D Systems)

Journal: bioRxiv

Article Title: Spatio-temporal dynamics of the fibrotic niche in cardiac repair

doi: 10.1101/2024.11.10.622609

Figure Lengend Snippet: A , Spatial maps showing MP and FB subtypes in the wound (LV edge in sham). B , day 7 neighborhoods surrounding Postn+Thbs4+ myoFB, with MP highlighted in blue. C , spatial gene factor co-variations of MP neighborhood in day 7 heart. Circle size scales linearly with −log 10 (p-value), and circle color indicates ridge regression coefficients. D , genes positively and negatively associated with gene factor 3 (Fa3) of MP and Fa2 of FB, respectively. Highlighted genes in MP-positive-Fa3: secreted ligands; MP-negative-Fa3: early proinflammatory/IFN pathway; FB-positive-Fa2: ECM genes, FB-negative-Fa2: cell cycle. E , pathways negatively associated with FB Fa2 (BioPlanet 2019 database). F , FB UMAP with inferred cell fate transition and cell cycle G2M gene score (upper), and time points post-lesion. G , boxplot comparing log2-transformed aggregated G2M gene expression of FB over time. H , CellChat ligand-receptor interaction prediction between MP (signal senders) and FB subtypes (signal receivers) across day 3–28. I , immune cell UMAP showing Gas6 and Pros1 expression, and FB UMAP showing Axl expression. J , multiplexed-IF staining of day 7 post-LAD IZ. Dotted lines highlight CD45+ GAS6+ PROS1+ immune cells contacting myoFB (CD140a+). K – M , mouse cardiac myoFB culturing experiment (Methods). Cultured myoFB were harvested for IF staining with αSMA and Mki67 ( K ), quantification of % Mki67+ nuclei (DAPI+) in αSMA+ cells (myoFB) ( l ), as well as qRT-PCR quantification of senescence marker gene Glb1 (β-gal) ( M ). N , CellChat prediction for FB (senders) and MP subtypes (receivers), across day 3-28. O , UMAPs of FB (left) and immune (right) clusters plotting SEMA3 family and their receptor’s gene expression. P and Q , mouse BMDM culturing. BMDM were exposed to BSA/SEMA3D for 72 hours, then harvested for H3P IF ( P ) with intensity quantification ( Q ). For ( L ), ( M ), ( P ), ( Q ), statistical significance was calculated by unpaired two-tailed t-test for two experimental groups. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; ns, not significant.

Article Snippet: The next day, the medium was removed and cells were washed twice with PBS, before being incubated with serum-free DMEM with Recombinant Mouse GAS6 protein (0.05– 1000ng/mL, R&D Systems, 986-GS-025/CF), Recombinant Mouse Protein S/PROS1 (100ng/mL, R&D Systems, 9740-PS-050/CF), or an equivalent amount of BSA.

Techniques: Transformation Assay, Expressing, Staining, Cell Culture, Quantitative RT-PCR, Marker, IF-P, Two Tailed Test

Journal: Transplantation

Article Title: Gas6/AXL Alleviates Hepatic Ischemia/Reperfusion Injury by Inhibiting Ferroptosis via the PI3K/AKT Pathway

doi: 10.1097/TP.0000000000005036

Figure Lengend Snippet: AXL activation attenuates lipid peroxidation in hepatic I/R injury in vivo. The mice underwent 1 h of liver ischemia followed by 6 h of reperfusion. AXL activator rmGas6 (5 μg), AXL inhibitor R428 (125 mg/kg), and PBS (vehicle) were intraperitoneally injected 2 h before liver ischemia. Mice were euthanized, and liver tissues and serums were collected for analysis. A, The area of liver necrosis was measured by H&E staining in the Sham and treatment mice after liver I/R injury. B, Serum levels of ALT and AST in Sham and I/R mice with or without rmGas6 or R428 treatment. C, Immunohistochemical analysis showing the expression of 4-HNE in liver tissues after I/R. D, MDA levels in liver tissues were determined by an assay kit. A–D, Each group n = 4–6. *** P < 0.001; ** P < 0.01; * P < 0.05. 4-HNE, 4-hydroxynonenal; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AXL, AXL receptor tyrosine kinase; Gas6, growth arrest-specific protein 6; I/R, ischemia/reperfusion, MDA, malondialdehyde.

Article Snippet: RmGas6 (R&D Systems, 8310-GS, United States), Fer-1 (MCE, HY-100579, United States), R428 (MCE, HY-15150, United States), and LY294002 (MCE, HY-10108, United States).

Techniques: Activation Assay, In Vivo, Injection, Staining, Immunohistochemical staining, Expressing

Journal: Transplantation

Article Title: Gas6/AXL Alleviates Hepatic Ischemia/Reperfusion Injury by Inhibiting Ferroptosis via the PI3K/AKT Pathway

doi: 10.1097/TP.0000000000005036

Figure Lengend Snippet: AXL activation alleviates iron accumulation to protect against hepatic I/R injury in vivo. A, Western blotting was used to assess the levels of the ferroptosis proteins TFR1, FTH1, ACSL4, and GPX4 after hepatic I/R injury in Sham and treatment groups. B and C, Iron stain and liver iron levels were examined by an assay kit after hepatic I/R injury in the Sham and rmGas6-treated group. D, TEM was used to detect the levels of mitochondrial damage after hepatic I/R injury in the Sham group and the treated group. A–D, Each group n = 4–6. *** P < 0.001; ** P < 0.01; * P < 0.05. ACSL4, acyl-CoA synthetase long-chain family member 4 antibody; AXL, AXL receptor tyrosine kinase; FTH1, ferritin heavy chain 1; Gas6, growth arrest-specific protein 6; GPX4, glutathione peroxidase 4; I/R, ischemia/reperfusion; p-AXL, phosphorylation AXL; rmGas6, recombinant mouse growth arrest-specific protein 6; TEM, transmission electron microscope; TFR1, transferrin receptor 1.

Article Snippet: RmGas6 (R&D Systems, 8310-GS, United States), Fer-1 (MCE, HY-100579, United States), R428 (MCE, HY-15150, United States), and LY294002 (MCE, HY-10108, United States).

Techniques: Activation Assay, In Vivo, Western Blot, Staining, Recombinant, Transmission Assay, Microscopy

Journal: Transplantation

Article Title: Gas6/AXL Alleviates Hepatic Ischemia/Reperfusion Injury by Inhibiting Ferroptosis via the PI3K/AKT Pathway

doi: 10.1097/TP.0000000000005036

Figure Lengend Snippet: AXL activation ameliorates hepatocyte ferroptosis in H/R injury. Mouse primary hepatocytes were exposed to hypoxia for 1 h followed by reoxygenation for 6 h and pretreated with the AXL activator rmGas6 or the AXL inhibitor R428 for 2 h before H/R. A, Western blotting was used to assess the levels of the ferroptosis factors TFR1, FTH1, ACSL4, and GPX4 after H/R in control and treated hepatocytes. B, ROS levels in hepatocytes were measured by upright fluorescence microscope. C, The levels of MDA in hepatocytes were assessed by an assay kit. D, The levels of Fe 2+ in hepatocytes. E, Representative immunofluorescence images of the GPX4 expression in hepatocytes exposed to H/R. F and G, The accumulation of lipid peroxidation in hepatocytes was analyzed by BODIPY staining, followed by immunofluorescence imaging and flow cytometry analysis, respectively. A–G, Each group n = 4–6. *** P < 0.001; ** P < 0.01; * P < 0.05. ACSL4, acyl-CoA synthetase long-chain family member 4 antibody; AXL, AXL receptor tyrosine kinase; BODIPY, boron dipyrromethene; DAPI, 4′,6-diamidino-2-phenylindole; FITC-A, fluorescein isothiocyanate-A; FTH1, ferritin heavy chain 1; Gas6, growth arrest-specific protein 6; GPX4, glutathione peroxidase 4; H/R, hypoxia/reoxygenation; MDA, malondialdehyde; p-AXL, phosphorylation AXL; rmGas6, recombinant mouse growth arrest-specific protein 6; ROS, reactive oxygen species; TFR1, transferrin receptor 1.

Article Snippet: RmGas6 (R&D Systems, 8310-GS, United States), Fer-1 (MCE, HY-100579, United States), R428 (MCE, HY-15150, United States), and LY294002 (MCE, HY-10108, United States).

Techniques: Activation Assay, Western Blot, Control, Fluorescence, Microscopy, Immunofluorescence, Expressing, Staining, Imaging, Flow Cytometry, Recombinant

Journal: Transplantation

Article Title: Gas6/AXL Alleviates Hepatic Ischemia/Reperfusion Injury by Inhibiting Ferroptosis via the PI3K/AKT Pathway

doi: 10.1097/TP.0000000000005036

Figure Lengend Snippet: AXL inhibition exacerbates ferroptosis in hepatic I/R injury through the PI3K/AKT pathway. A, Western blot analysis of the protein expression of PI3K, p-PI3K, AKT, and p-AKT in liver tissue in the Sham group, the rmGas6-treated group and the R428 treatment group after liver I/R. B, Western blot analysis of the expression of PI3K, p-PI3K, AKT, and p-AKT in the control group, the rmGas6-treated and the R428-treated group after H/R. C, Serum levels of ALT and AST in hepatic I/R-subjected mice treated with or without the LY294002. D, H&E staining was used to detect liver injury in Gas6-treated or LY294002-treated mice after hepatic I/R injury. E, Western blot analysis of PI3K, p-PI3K, AKT, p-AKT, TFR1, FTH1, ACSL4, and GPX4 expression in Sham and treatment groups after hepatic I/R injury. F, Western blot analysis of PI3K, p-PI3K, AKT, p-AKT, TFR1, FTH1, ACSL4, and GPX4 expression in the control and treatment primary hepatocytes after H/R injury. A–F, Each group n = 4–6. *** P < 0.001; ** P < 0.01; * P < 0.05. ACSL4, acyl-CoA synthetase long-chain family member 4 antibody; AKT, the Ser and Thr kinase AKT; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AXL, AXL receptor tyrosine kinase; FTH1, ferritin heavy chain 1; Gas6, growth arrest-specific protein 6; GPX4, glutathione peroxidase 4; H&E, hematoxylin and eosin; H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; p-AKT, phospho-AKT; PI3K, phosphatidylinositol 3-kinase; p-PI3K, phosphorylation phosphatidylinositol 3-kinase; rmGas6, recombinant mouse growth arrest-specific protein 6; TFR1, transferrin receptor 1.

Article Snippet: RmGas6 (R&D Systems, 8310-GS, United States), Fer-1 (MCE, HY-100579, United States), R428 (MCE, HY-15150, United States), and LY294002 (MCE, HY-10108, United States).

Techniques: Inhibition, Western Blot, Expressing, Control, Staining, Recombinant

Journal: Cell Biology and Toxicology

Article Title: Administration of Gas6 attenuates lung fibrosis via inhibition of the epithelial-mesenchymal transition and fibroblast activation

doi: 10.1007/s10565-024-09858-5

Figure Lengend Snippet: Inhibition of EMT and primary ATII cell invasion by rGas6 administration. Mice were intratracheally instilled with BLM (5 U/kg). Either rGas6 (50 μg/kg) or saline (Sal) was intraperitoneally administered 1 day before BLM treatment and once every 2 days thereafter. Mice were euthanized 14 days after BLM treatment. ( a ) Left: morphological changes in isolated ATII cells (Scale bars: 100 μm). Representative images are shown from three replicates per condition with cells pooled from two mice per replicate. Right: percentage of spindle shaped cells/high-power fields (HPF). ( b ) qRT-PCR of EMT markers in ATII cell samples. ( c ) Left: immunofluorescence staining for E-cadherin (green) and α-SMA (red). Right: quantification of proteins in ATII cells. Original magnification: 400 × . Scale bars: 20 μm. Imaging medium: Vectashield fluorescent mounting medium containing DAPI. ( d ) Immunoblot analysis of E-cadherin and N-cadherin in lung homogenates. Below: Densitometric analysis of each band normalized to that of β-actin. Values represent the means ± S.E.M. from three mice per group. ( e ) qRT-PCR of Snail1, Zeb1, and Twist1 in ATII cell samples. ( f ) Phase-contrast microscopy and quantification of invaded ATII cells. Scale bars: 100 µm. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with control or for BLM + Sal vs. BLM + rGas6. Data were obtained from three ( c right , f below ) or five replicates ( b , e ) per condition with cells pooled from two mice per replicate. Data are shown as the means ± S.E.M. ( g ) Immunofluorescence staining for E-cadherin (red), α-SMA (red), or S100A4 (green) in lung sections. Arrowheads indicate colocalization of E-cadherin in lung fibroblasts. Imaging medium: Vectashield fluorescence mounting medium containing DAPI. Scale bars: 20 μm. Representative images were obtained from three mice in each group. ( h ) Graph representing the number of S100A4/E-cadherin double-positive cells compared with the total S100A4-positive cell population in lung parenchyma. Mean of five HPFs/section ± S.E.M. from three mice in each group

Article Snippet: Mouse rGas6 (986-GS) was acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Inhibition, Saline, Isolation, Quantitative RT-PCR, Immunofluorescence, Staining, Imaging, Western Blot, Microscopy, Control, Fluorescence

Journal: Cell Biology and Toxicology

Article Title: Administration of Gas6 attenuates lung fibrosis via inhibition of the epithelial-mesenchymal transition and fibroblast activation

doi: 10.1007/s10565-024-09858-5

Figure Lengend Snippet: Inhibition of apoptosis in ATII cells by rGas6 administration. The experimental design was as described in Fig. . Mice were euthanized 14 days after BLM treatment. ( a ) Left: Representative TUNEL-stained and fixed ATII cells (original magnification: 400 ×). Positive staining depicted in green. Nuclei were observed by DAPI staining. Scale bars: 20 μm. Right: Quantitation of the number of TUNEL-positive cells (number/HPF) in the different groups. ( b ) The cell viability in primary ATII cells was measured by flow cytometry after annexin V-FITC/PI dual staining. Apoptotic cells were quantified as the sum of the percentages of cells in the early and late stages of apoptosis. ** P < 0.01, *** P < 0.001 compared with control or for BLM + Sal vs. BLM + rGas6. Data were obtained from three replicates per condition with cells pooled from two mice per replicate ( a right , b right ). The data are shown as the means ± S.E.M. ( c ) Immunoblot analysis of Bax, Bcl-2, cleaved caspase-3, and cleaved PARP in lung homogenates. Below: Densitometric analysis of each band normalized to that of β-actin. ( d ) Representative confocal images of lung sections stained with an anti-SPC antibody (red), anti-cleaved caspase-3 antibody (green), and DAPI (blue) ( left ). Original magnification: × 400. Scale bars = 20 μm. Quantification of cleaved caspase-3 staining in SPC. + ATII cells ( right ). The values represent the means ± S.E.M. of results from three mice from each group. ** P < 0.01 compared with Sal control or for BLM + Sal vs. BLM + rGas6

Article Snippet: Mouse rGas6 (986-GS) was acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Inhibition, TUNEL Assay, Staining, Quantitation Assay, Flow Cytometry, Control, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Administration of Gas6 attenuates lung fibrosis via inhibition of the epithelial-mesenchymal transition and fibroblast activation

doi: 10.1007/s10565-024-09858-5

Figure Lengend Snippet: Inhibition of fibroblast activation by rGas6 administration. The experimental design was as described in Fig. . Mice were euthanized 14 ( a , d-g ) or 21 days ( b ) after BLM treatment. ( a , d–f ) Primary fibroblasts were isolated from murine lungs. ( a ) qRT-PCR of collagen type 1, fibronectin, and α-SMA in fibroblast samples. ( b ) Immunofluorescence staining for α-SMA (red) or S100A4 (green) was performed in lung sections. Arrowheads indicate colocalization of α-SMA in lung fibroblasts. Imaging medium: Vectashield fluorescence mounting medium containing DAPI. Scale bars: 20 μm. Representative images were obtained from three mice per group. ( c ) Graph representing the number of S100A4/α-SMA double-positive cells compared with the total S100A4-positive cell population in the lung parenchyma. Mean of five HPFs per section ± S.E.M. from three mice in each group. *** P < 0.001 compared with control or for BLM + Sal vs. BLM + rGas6. ( d ) Phase-contrast microscopy ( left ) and quantification of invaded fibroblasts ( right ) using Matrigel-coated Transwell plates. Scale bar: 100 µm. ( e ) qRT-PCR of Has2 , CD44 , MMP9, MMP12, and MMP14 in fibroblast samples. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with control or for BLM + Sal vs. BLM + rGas6. Data were obtained from five replicates per condition with cells pooled from two mice per replicate ( a , d right , e ). The data are shown as the means ± S.E.M. ( f ) Selected heatmaps showing differentially expressed genes encoding adhesion and ECM molecules in primary lung fibroblasts between the BLM + Sal and BLM + rGas6 groups. Red: increased expression; blue: decreased expression. Data were obtained from two replicates per condition with cells pooled from two mice per replicate. ( g ) Relative expression levels of selected genes from PCR array profiling ( f ). Log 2 fold-change values (ApoSQ-CAF CM vs. CAF CM, fold change > 1.5)

Article Snippet: Mouse rGas6 (986-GS) was acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Inhibition, Activation Assay, Isolation, Quantitative RT-PCR, Immunofluorescence, Staining, Imaging, Fluorescence, Control, Microscopy, Expressing

Journal: Cell Biology and Toxicology

Article Title: Administration of Gas6 attenuates lung fibrosis via inhibition of the epithelial-mesenchymal transition and fibroblast activation

doi: 10.1007/s10565-024-09858-5

Figure Lengend Snippet: Inhibition of lung fibrosis by rGas6 administration. The experimental design was as described in Fig. . Mice were euthanized on days 14 and 21 after BLM treatment. ( a , b ) Levels of the active form of the TGF-β1 and HGF proteins in BAL fluid were quantified by ELISAs. ( c ) qRT-PCR of collagen type1, fibronectin, and α-SMA in lung tissue samples. ( d ) Left: Immunoblot analysis of the indicated proteins in lung homogenates. Right: Densitometric analysis of each band normalized to that of β-actin. ( e ) Collagen deposition in the whole lung was determined by measuring the hydroxyproline content on day 21. ( f ) Lung sections were visualized with Masson’s trichrome staining on day 21. Representative results from five mice per group are shown (scale bar: 50 μm). ( g ) Ashcroft scoring of the lung sections. The values represent the means ± S.E.M. of results from three ( d ) or five mice ( a – c , e , g ) in each group. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with Sal control or for BLM + Sal vs. BLM + rGas6

Article Snippet: Mouse rGas6 (986-GS) was acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Inhibition, Quantitative RT-PCR, Western Blot, Staining, Control

Journal: Cell Biology and Toxicology

Article Title: Administration of Gas6 attenuates lung fibrosis via inhibition of the epithelial-mesenchymal transition and fibroblast activation

doi: 10.1007/s10565-024-09858-5

Figure Lengend Snippet: Axl activation and COX-2-derived PGE 2 and PGD 2 production induced by rGas6 administration. The experimental design was as described in Fig. . Mice were euthanized on day 14 after BLM treatment. ( a ) Left: Immunofluorescence staining for phospho-Axl (green), total Axl (red), phospho-Mer (red), and total Mer (green) in primary ATII cells. Images were captured at 400 × magnification. Right: Quantification of phospho-Axl, total Axl, phospho-Mer, and total Mer staining in ATII cells. Imaging medium: Vectashield fluorescence mounting medium containing DAPI. Scale bars: 20 μm. Data were obtained from three replicates per condition with cells pooled from two mice per replicate. ( b ) Left: Immunoblot analysis of total/phospho-Axl and total/phospho-Mer in lung tissue homogenates. Right: Densitometric analysis of each band normalized to that of β-actin. ( c ) Immunoblot analysis of total/phospho-Akt in lung tissue homogenates. Below: Densitometric analysis of each band normalized to that of total Akt. Data are from independent experiments with three mice per group (mean ± S.E.M.). ( d , e ) qRT-PCR of COX-2 and COX-1 in ATII cells and lung tissue samples. ( f ) Immunoblot analysis of COX-2 and COX-1 in lung tissue homogenates. Below: Densitometric analysis of each band normalized to that of β-actin. ( d ) Data were obtained from five replicates per condition with cells pooled from two mice per replicate. Data were obtained from independent experiments with five ( e ) or three ( f ) mice per group. ( g , h ) PGE 2 or PGD 2 levels in BAL fluid (BALF, n = 5 mice) and culture supernatants from ATII cells and alveolar macrophages (AM) were measured using an enzyme immunoassay. ( h ) Data were obtained from five replicates per condition with cells pooled from two mice per replicate. Values represent the means ± S.E.M. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with Sal control or for BLM + Sal vs. BLM + rGas6

Article Snippet: Mouse rGas6 (986-GS) was acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Derivative Assay, Immunofluorescence, Staining, Imaging, Fluorescence, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell Biology and Toxicology

Article Title: Administration of Gas6 attenuates lung fibrosis via inhibition of the epithelial-mesenchymal transition and fibroblast activation

doi: 10.1007/s10565-024-09858-5

Figure Lengend Snippet: Inhibition of EMT and fibroblast activation via Gas6/Axl signaling events. Where indicated, the Axl inhibitor BGB324 (BGB, 5 mg/kg, i.o. ), COX-2 inhibitor NS-398 (NS, 5 mg/kg, i.o. ), EP1/EP2 inhibitor AH-6809 (AH, 5 mg/kg, i.p. ), or DP2 inhibitor BAY-u3405 (BAY, 30 mg/kg, i.p. ) was co-administered with rGas6 1 day before BLM treatment and then administered once/day (AH) or once every 2 days (BGB, NS, and BAY). Mice were euthanized 14 days following BLM treatment. ( a , b ) qRT-PCR of EMT markers and EMT-regulating transcription factors in primary ATII cells. ( c , d ) qRT-PCR of activated fibroblast markers and invasive myofibroblast-related molecules in primary lung fibroblasts. ( e ) Left: The cells were visualized by phase-contrast microscopy to analyze their invasive ability in Matrigel-coated Transwell assays. Scale bar: 100 µm. Right: The invaded fibroblasts were quantified by counting the number of cells adhering to the bottom surface of the upper chamber. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with BLM + Sal or for BLM + Gas6 vs. BLM + rGas6 + the inhibitor. Data were obtained from five replicates per condition with cells pooled from three mice per replicate (means ± S.E.M.)

Article Snippet: Mouse rGas6 (986-GS) was acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Inhibition, Activation Assay, Quantitative RT-PCR, Microscopy

Journal: Cell Biology and Toxicology

Article Title: Administration of Gas6 attenuates lung fibrosis via inhibition of the epithelial-mesenchymal transition and fibroblast activation

doi: 10.1007/s10565-024-09858-5

Figure Lengend Snippet: A schematic diagram summarizing the role of Gas6/Axl signaling events for the prevention of lung fibrosis. rGas6 inhibits EMT and apoptosis in ATII cells and concomitantly suppresses fibroblast activation, consequently preventing the development of BLM-induced lung fibrosis. This occurs through the activation of Axl signaling pathway, including COX-2-derived PGE 2 and PGD 2 production in ATII cells and alveolar macrophages

Article Snippet: Mouse rGas6 (986-GS) was acquired from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Derivative Assay