Journal: bioRxiv

Article Title: Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis

doi: 10.1101/2022.09.20.508661

Figure Lengend Snippet: (A) Immunofluorescence staining for F4/80 (red) and GAS6 (green) in synovial tissue from non-obese, non-obese OA, obese, and obese OA patients. Scale bar: 50 μm. (B) Quantification of F4/80-GAS6-positive macrophages as a proportion of total lining cell population in (A), n=6 per group. (C) Immunofluorescence staining (first line) for F4/80 (red) and GAS6 (green) in synovial tissue and immunohistochemical staining of GAS6 (second line) in cartilage of controls and DMM from C57BL/6 and ApoE −/− mice. Scale bar: 50 μm. (D) Quantification of F4/80-GAS6 positive macrophages (yellow) as a proportion of total F4/80 positive cells in (C) (first line). Quantification of GAS6-positive cells in (C) (second line), n=6 per group. (E) Enzyme-linked immunosorbent assay (ELISA) for GAS6 in synovial fluid of non-obese and obese OA patients, n=13 per group. (F) Immunofluorescence staining for F4/80(red) and GAS6 (green) in RAW264.7 cells treated with LPS for 24 and 48 h. Scale bar: 50 μm; (G) Quantification of F4/80-GAS6-positive macrophages (yellow) as a proportion of total F4/80-positive cells (red), n=6 per group. * P<0.05, ** P<0.01, *** P<0.001, NS=not significant. One-way analysis of variance (ANOVA) was performed. Data are shown as mean ± SD.

Article Snippet: After the surgery, 50 ng/g of recombinant mouse GAS6 (rmGAS6, Sino Biological, China, #58026-M08H) was administered into the articular once per week.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis

doi: 10.1101/2022.09.20.508661

Figure Lengend Snippet: (A) Differentially-expressed mRNA in bone marrow-derived macrophages from normal controls or lipopolysaccharide treatment based on GSE53986. (B) Relative mRNA expression level of GAS6 in LPS-treated RAW264.7 cells, n=3 per group. (C) Relative mRNA expression level of IL-1β, IL-6, and TNF-α in LPS and rmGAS6-treated RAW264.7 cells, n=3 per group. (D) Immunofluorescence of F4/80 (red) and AXL (green) in synovial tissue from non-obese human, non-obese OA patients, obese human, and obese OA patients. Scale bar: 50 μm. (E) Immunofluorescence staining of F4/80 (red) and AXL (green) in synovial tissue in controls and DMM cartilage from C57BL/6 and ApoE −/− mice. Scale bar: 50 μm; (F and G) Quantification of F4/80-AXL-positive macrophages (yellow) as a proportion of total F4/80-positive cells in (D and E), n=6 per group. *P<0.05, **P<0.01, NS=not significant. One-way analysis of variance (ANOVA) was performed. Data are shown as mean ± SD.

Article Snippet: After the surgery, 50 ng/g of recombinant mouse GAS6 (rmGAS6, Sino Biological, China, #58026-M08H) was administered into the articular once per week.

Techniques: Derivative Assay, Expressing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis

doi: 10.1101/2022.09.20.508661

Figure Lengend Snippet: (A) Immunofluorescence staining for caspase-3 (top) and TUNEL (lower) in normal and OA synovial tissue from non-obese and obese patients. Scale bar: 50 μm. Quantification of caspase-3- or TUNEL-positive cells as a proportion of total lining cell population in (A), n=6 per group. (C) Immunofluorescence staining for caspase-3 (top) and TUNEL (lower) in controls and DMM synovial tissue from C57 and ApoE−/− mice. Scale bar: 50 μm. (D) Quantification of caspase-3- or TUNEL-positive cells as a proportion of lining cell population in (C), n=6 per group. (E) Immunofluorescence staining for F4/80 (red) in BMDMs extracted from ApoE−/− and C57BL/6 mice. CFSE (green) in apoptotic thymocytes of C57BL/6 mice after 2-h phagocytosis. (F) Quantification of positive BMDMs engulfing apoptotic thymocytes as a proportion of total F4/80-positive cells, n=5 per group. (G) mRNA expression levels of iNOS or Arg1 after LPS, rmGAS6, or IL-4 stimulation for 24 h. (H) Immunofluorescence staining for F4/80 (red) in RAW264.7 cells and CFSE (green) in apoptotic thymocytes after phagocytosis for 2 h. Scale bar: 50 μm. Quantification of positive RAW264.7 cells engulfing apoptotic thymocytes as a proportion of total F4/80-positive cells, n=5 per group. (I) Flow cytometry analysis of CFSE-positive cells in total macrophages is shown as fluorescence-intensity distribution plots. (J) Efferocytotic index was calculated as percentage of CFSE-positive cells divided by percentage of total cells, n=6 per group. *P<0.05, **P<0.01, ***P<0.001, and NS=not significant. One-way analysis of variance (ANOVA) was performed. Data are shown as mean ± SD.

Article Snippet: After the surgery, 50 ng/g of recombinant mouse GAS6 (rmGAS6, Sino Biological, China, #58026-M08H) was administered into the articular once per week.

Techniques: Immunofluorescence, Staining, TUNEL Assay, Expressing, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis

doi: 10.1101/2022.09.20.508661

Figure Lengend Snippet: (A) Safranin O and Fast Green staining (top and middle) of knee cartilage, H&E staining of synovial tissues from DMM mice and DMM mice treated with R428, and ApoE −/− mice treated with recombinant mouse (rmGAS6) eight weeks after surgery. Scale bar: 200 μm, 50 μm. (B) Quantitative analysis of Osteoarthritis Research Society International (OARSI) scale and synovitis score in (A), n=6 per group. (C) Immunofluorescence staining of caspase-3 or TUNEL in synovial tissue from DMM mice, DMM mice treated with R428, and ApoE −/− mice treated with recombinant mouse (rmGAS6) eight weeks after surgery. Scale bar: 50 μm. (D) Quantification of caspase-3- or TUNEL-positive cells as a proportion of lining cell population in (C), n=6 per group. *P<0.05, **P<0.01, ***P<0.001, NS=not significant. One-way analysis of variance (ANOVA) was performed. Data are shown as mean ± SD.

Article Snippet: After the surgery, 50 ng/g of recombinant mouse GAS6 (rmGAS6, Sino Biological, China, #58026-M08H) was administered into the articular once per week.

Techniques: Staining, Recombinant, Immunofluorescence, TUNEL Assay

Journal: bioRxiv

Article Title: Down-regulated GAS6 impairs synovial macrophage efferocytosis and promotes obesity-associated osteoarthritis

doi: 10.1101/2022.09.20.508661

Figure Lengend Snippet: Macrophage polarization induced by obesity decreased the secretion of GAS6 and impaired the phagocytosis of apoptotic cells. The accumulation of apoptotic cell debris lead to the persistence of local inflammation and synovial hyperplasia, which aggravates the pathological process of OA.

Article Snippet: After the surgery, 50 ng/g of recombinant mouse GAS6 (rmGAS6, Sino Biological, China, #58026-M08H) was administered into the articular once per week.

Techniques:

Journal: Scientific Reports

Article Title: Axl is required for TGF-β2-induced dormancy of prostate cancer cells in the bone marrow

doi: 10.1038/srep36520

Figure Lengend Snippet: ( A ) mRNA expression in control and Gas6 overexpressed PCa cells. mRNA was extracted 2 days after serum starvation. *p < 0.05, **p < 0.01 compared to PC3 Control cells. ## p < 0.01 compared to DU145 Control cells. ( B ) mRNA expression in sh control and sh Gas6 knockdown PCa cells. mRNA was extracted after 6 days of culture in 0.5% FBS. Culture media was replaced at day3.*p < 0.05, **p < 0.01 compared to PC3 sh Control cells. ## p < 0.01 compared to DU145 sh Control cells. ( C–E ) mRNA expression in PCa cells 6 days after siRNA treatments targeting Axl. *p < 0.05, **p < 0.01 compared to si scramble control PC3cells, ## p < 0.01 compared to si scramble control DU145 cells. ( F,G ) Western blots showing protein expression levels of Axl, TGFBR2 and TGFBBR3 in PCa cells after the reduction of Axl or Gas6 by shRNA ( F ) or siRNA ( G ). Protein samples were loaded 6 days after siRNA treatments targeting Axl (G) .

Article Snippet: An antibody sandwich ELISA was used to evaluate Gas6 expression in the conditioned media and cell lysates by the following the directions of manufacturer (Cat. #DY986 and DY885, R&D Systems, Minneapolis, MN).

Techniques: Expressing, Control, Knockdown, Western Blot, shRNA

Journal: Scientific Reports

Article Title: Axl is required for TGF-β2-induced dormancy of prostate cancer cells in the bone marrow

doi: 10.1038/srep36520

Figure Lengend Snippet: ( A ) Relative mRNA levels in PCa cells were evaluated 24 hours after TGF-β1 (Cat #: 240-B/CF, R&D Systems, Minneapolis, MN) or TGF-β2 (Cat #: 302-B2/CF, R&D Systems, Minneapolis, MN) stimulation (both 2 ng/ml). *p < 0.05, **p < 0.01 compared to PCa cells without TGF-β stimulation, # p < 0.05, ## p < 0.01 compared to PCa with TGF-β1 stimulation. ( B,C ) PCa cells were pre-labeled with DiD, and DiD retention was evaluated 6 days after TGF-β2 stimulation by flow cytometry. *p < 0.05, **p < 0.01 compared to PC3 cells without TGF-β2 treatments, ## p < 0.01 compared to DU145 cells without TGF-β2 treatments in each of sh Control, sh Axl or sh Gas6 cells. ( D ) Western blots showing p27 expression levels in PCa cells 3 days after TGF-β2 treatment. ( E ) Relative p27 expression levels evaluated by densitometry. P27 expression was normalized by β-actin expression. *p < 0.05, **p < 0.01 compared to PCa sh Control cells without TGF-β2 treatments. # p < 0.05, ## p < 0.01 compared to DU145 sh Axl cells without TGF-β2 stimulation.

Article Snippet: An antibody sandwich ELISA was used to evaluate Gas6 expression in the conditioned media and cell lysates by the following the directions of manufacturer (Cat. #DY986 and DY885, R&D Systems, Minneapolis, MN).

Techniques: Labeling, Flow Cytometry, Control, Western Blot, Expressing

Journal: Scientific Reports

Article Title: Axl is required for TGF-β2-induced dormancy of prostate cancer cells in the bone marrow

doi: 10.1038/srep36520

Figure Lengend Snippet: Gas6 produced by osteoblasts binds to Axl expressed by disseminated PCa cells, and its signaling induces expression of both TGF-β ligands (TGF-β1 and TGF-β2) and their receptors (TGFBR2 and TGFBR3). Subsequently, autocrine and paracrine TGF-β signaling induces PCa dormancy.

Article Snippet: An antibody sandwich ELISA was used to evaluate Gas6 expression in the conditioned media and cell lysates by the following the directions of manufacturer (Cat. #DY986 and DY885, R&D Systems, Minneapolis, MN).

Techniques: Produced, Expressing

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: bioRxiv

Article Title: Gas6 ameliorates intestinal mucosal immunosenescence to prevent the translocation of a gut pathobiont, Klebsiella pneumoniae , to the liver

doi: 10.1101/2023.01.19.524842

Figure Lengend Snippet: (A) Cytokine array analysis of the K. pneumoniae infection model based on the Transwell insert co-culture system consisting of Caco-2 cells and RAW264.7 macrophages. Quantification of Axl signals using a laser scanner. Data are presented as the mean ± SD. ** p < 0.01. (B and C) Secretion of Axl (B) and Gas6 (C) by Caco-2 cells or RAW264.7 macrophages. Culture supernatants or lysates of K. pneumoniae were added to Caco-2 cells or RAW264.7 macrophages for 12 h. The culture media were then collected for ELISA analysis of Axl or Gas6 levels. Data are presented as the mean ± SD. ** p < 0.01. (D) K. pneumoniae -infected Caco-2 cells grown on a Transwell insert in the presence or absence of RAW264.7 macrophages were immunostained with anti-Gas6 and anti-Axl antibodies. Scale bar = 50 μm.

Article Snippet: An Axl inhibitor (R428) (Abcam, Cambridge, UK, cat# ab141364), recombinant human Gas6 protein (R&D Systems, Minneapolis, MN, USA, cat# 885-GSB), recombinant mouse Gas6 protein (R&D Systems, cat# 986-GS) (used in administration experiments in mice), and an anti-Gas6 antibody (R&D Systems, cat# AF986) were used as Gas6/Axl signal modulators.

Techniques: Infection, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Gas6 ameliorates intestinal mucosal immunosenescence to prevent the translocation of a gut pathobiont, Klebsiella pneumoniae , to the liver

doi: 10.1101/2023.01.19.524842

Figure Lengend Snippet: (A) Western blotting was performed to detect the expression of Axl, Gas6, ZO-1, and occludin in Caco-2 cells infected with K. pneumoniae in the presence of an Axl inhibitor (R428) or an anti-Gas6 antibody. Prior to K. pneumoniae infection, cells were treated for 3 h with Axl inhibitor (R428; 20 nM) or 1 μg of anti-Gas6 antibody. (B and C) Axl inhibitor (R428; 20 nM) or 1 μg of anti-Gas6 antibody were added to Caco-2 cells grown on the insert in in the presence or absence of RAW264.7 macrophages for 3 h prior to K. pneumoniae infection. Next, Caco-2 cells were immunostained with an anti-ZO-1 antibody (B), an anti-occludin antibody (B), and an anti- Klebsiella pneumoniae antibody (C). Scale bar = 50 μm. Immunostaining intensities of ZO-1 (B), occludin (B), and K. pneumoniae (C) were analyzed by ImageJ software. Data are presented as the mean ± SD. ** p < 0.01.

Article Snippet: An Axl inhibitor (R428) (Abcam, Cambridge, UK, cat# ab141364), recombinant human Gas6 protein (R&D Systems, Minneapolis, MN, USA, cat# 885-GSB), recombinant mouse Gas6 protein (R&D Systems, cat# 986-GS) (used in administration experiments in mice), and an anti-Gas6 antibody (R&D Systems, cat# AF986) were used as Gas6/Axl signal modulators.

Techniques: Western Blot, Expressing, Infection, Immunostaining, Software

Journal: bioRxiv

Article Title: Gas6 ameliorates intestinal mucosal immunosenescence to prevent the translocation of a gut pathobiont, Klebsiella pneumoniae , to the liver

doi: 10.1101/2023.01.19.524842

Figure Lengend Snippet: (A and B) Sections of cecal mucosa (A) or liver (B) were from mice aged 15 or 57 weeks at 2 days after infection with K. pneumoniae ATCC43816 pmCherry and immunostained with an anti-Gas6 antibody and an anti-Axl antibody. Scale bar = 50 μm. (C) Detection of Axl, Gas6, ZO-1, and occludin by western blotting. Cecum and liver tissues were collected, homogenized, and analyzed using an anti-Axl antibody, an anti-Gas6 antibody, an anti-ZO-1 antibody, or an anti-occludin antibody. Signal intensity was analyzed by ImageJ software. Data are presented as the mean ± SD (n = 5 per group). NS: not significant, * p < 0.05, ** p < 0.01. (D) Linear correlation between Gas6 expression and Axl expression in the cecum or liver of mice aged 15 (red circles) or 57 (blue circles) weeks infected with K. pneumoniae ATCC43816 pmCherry. r > 0.70 and p < 0.05. (E) The population of CD11b+F4/80+ macrophages in the intestinal mucosa of young (14-week-old) and old (56-week-old) mice was examined by staining with anti-F4/80 and anti-CD11b antibodies. The percentage of F4/80-positive/CX3CR1-negative cells in 14-week-old and 56-week-old mice was 29.5% and 8.87%, respectively. Data are representative of four mice.

Article Snippet: An Axl inhibitor (R428) (Abcam, Cambridge, UK, cat# ab141364), recombinant human Gas6 protein (R&D Systems, Minneapolis, MN, USA, cat# 885-GSB), recombinant mouse Gas6 protein (R&D Systems, cat# 986-GS) (used in administration experiments in mice), and an anti-Gas6 antibody (R&D Systems, cat# AF986) were used as Gas6/Axl signal modulators.

Techniques: Infection, Western Blot, Software, Expressing, Staining

Journal: bioRxiv

Article Title: Gas6 ameliorates intestinal mucosal immunosenescence to prevent the translocation of a gut pathobiont, Klebsiella pneumoniae , to the liver

doi: 10.1101/2023.01.19.524842

Figure Lengend Snippet: (A) Administration of Gas6 recombinant protein to Caco-2 cells to analyze the expression of Axl, ZO-1, and occludin. Addition of Gas6 recombinant protein to the apical surface (left scheme) or the basolateral side (right scheme) of Caco-2 cells. (B) Western blot analysis to detect Axl, Gas6, ZO-1, and occludin in Caco-2 cells treated with human Gas6 recombinant protein from the apical or basolateral sides. (C) Western blot analysis was performed to detect Axl, Gas6, ZO-1, and occludin. Gas6 recombinant protein (1 μg) was added to the apical side of Caco-2 cells grown in a Transwell co-culture system for 3 h prior to K. pneumoniae infection. (D and E) Gas6 recombinant protein (1 μg) was added to Caco-2 cells grown on the Transwell insert for 3 h prior to K. pneumoniae infection. Next, Caco-2 cells were immunostained with an anti-ZO-1 antibody (C), an anti-occludin antibody (C), an anti- Klebsiella pneumoniae antibody (D), and an anti-E-cadherin antibody. The signal intensities of ZO-1, occluding, E-cadherin, and K. pneumoniae were analyzed by ImageJ software. Data are presented as the mean ± SD. NS: not significant, ** p < 0.01.

Article Snippet: An Axl inhibitor (R428) (Abcam, Cambridge, UK, cat# ab141364), recombinant human Gas6 protein (R&D Systems, Minneapolis, MN, USA, cat# 885-GSB), recombinant mouse Gas6 protein (R&D Systems, cat# 986-GS) (used in administration experiments in mice), and an anti-Gas6 antibody (R&D Systems, cat# AF986) were used as Gas6/Axl signal modulators.

Techniques: Recombinant, Expressing, Western Blot, Co-Culture Assay, Infection, Software

Journal: bioRxiv

Article Title: Gas6 ameliorates intestinal mucosal immunosenescence to prevent the translocation of a gut pathobiont, Klebsiella pneumoniae , to the liver

doi: 10.1101/2023.01.19.524842

Figure Lengend Snippet: (A) Treatment scheme used to analyze the effect of Gas6 recombinant protein on the susceptibility of 57-week-old mice to infection by K. pneumoniae . Antibiotics were administered 4 weeks before administration of Gas6 recombinant protein. Gas6 recombinant protein (125 μg protein/kg) was administered intraperitoneally to 57-week-old mice three times every 24 h prior to bacterial infection. (B) Effect of Gas6 recombinant protein on survival of 57-week-old mice infected with K. pneumoniae ATCC43816 pmCherry. Each mouse was orally inoculated with K. pneumoniae ATCC43816 pmCherry (5 × 10 7 bacteria). p -values were determined using the log-rank test (n = 6 per group). (C) Bacterial counts in cecum and liver tissues were determined 2 days post-infection. Cecum and liver tissues were homogenized in PBS. The homogenates were plated on LB agar containing 400 μg/mL ampicillin and the number of CFU was counted. Data are presented as the mean ± SD. (n = 6 per group). * p < 0.05, ** p < 0.01. (D) Detection of Axl, Gas6, ZO-1, and occludin by western blotting. Cecum tissues were collected, homogenized, and analyzed by western blotting with anti-Axl, anti-Gas6, anti-ZO-1, or anti-occludin antibodies. Signal intensity was analyzed by ImageJ software. Data are presented as the mean ± SD (n = 5 per group). NS: not significant, * p < 0.05, ** p < 0.01.

Article Snippet: An Axl inhibitor (R428) (Abcam, Cambridge, UK, cat# ab141364), recombinant human Gas6 protein (R&D Systems, Minneapolis, MN, USA, cat# 885-GSB), recombinant mouse Gas6 protein (R&D Systems, cat# 986-GS) (used in administration experiments in mice), and an anti-Gas6 antibody (R&D Systems, cat# AF986) were used as Gas6/Axl signal modulators.

Techniques: Recombinant, Infection, Western Blot, Software

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