Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 1. Differentiation of THP-1 cells induced by PMA. (A) THP-1 monocytes are differentiated to macrophages and induced to express Mertk by the addition of 100 ng/mL of PMA for at least 24 h, while Axl expression is not induced. Likewise, Tyro3 expression remains stable and is unaffected by treatment. H1299 and Beas2B cells are used as positive controls for Mertk/Axl and Tyro3, respec- tively. The observed doublet for Mertk corresponds to fully glycosylated and partially glycosylated glycoforms. (B) THP-1 cells treated with 100 ng/mL of PMA over 48 h exhibit a decreased expression of Gas6 but an increased expression of Protein S. (C) MertK and Gas6 expression patterns over a time of 48 h after 100 ng/mL of PMA treatment as quantified from Western blots. (D) Bar plots showing MerTK and Gas6 expression quantified from Western blots at 0 h and post 72 h of 100 ng/mL of PMA treatment (significant differences were observed between the 0 h and 72 h time points for Gas6 (* p = 0.029) and MerTK (** p = 0.003), as determined by independent t-tests). (E) THP-1 cells were treated with 100 ng/mL of PMA over 72 h. mRNA was analyzed via qRT-PCR for Mertk, Axl, Protein S (ProS), Tyro3, and Gas6. Mertk transcription was highly elevated, more than 10-fold, due to PMA treatment, while Gas6 transcription was repressed more than 10-fold. (F) PMA treatment of THP-1s illustrates a complimentary phenomenon. As a monocyte, Gas6 is expressed with little Mertk expression; however, when differentiated, Mertk is highly expressed in favor of Gas6.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 3. γ-carboxylated Gas6 Reduces Full-length Mertk Expression and Decreases sMertk. (A) γ- carboxylation status (Gla) of Gas6 is regulated with the addition of Vitamin K or warfarin, producing either a γ-carboxylated, active ligand or a non-γ-carboxylated, inactive ligand, respectively. The carboxylation status (Gla) domain of Gas6 can bind with externalized PS. (B) Recombinant inactive (Gas6-W) and active (Gas6-VK) were produced via HEK293 transfection, with a mock transfection control (Mock). The amount of Gas6 was observed (bottom), while the carboxylation status that is responsible for ligand activity was determined for each (left, top). (C) PMA-differentiated THP-1s were treated for 4 h with either serum-free RPMI only (-), a mock transfection control, 10 nM of active Gas6 (Gas6-VK), or 10 nM of inactive Gas6 (Gas6-W). Treatments were alone or combined with inhibitors of 3 µM of GW280264X (an ADAM17 inhibitor), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a proteasomal inhibitor). (D) Quantitative results indicate that Gas6, either active or inactive, does not stabilize the C-terminal fragments as shown in the DAPT and MG132 treatments (bands at 75 kDa). As denoted by sMertk (top), the cleavage is decreased with GW280264X treatment compared to the untreated control.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Expressing, Recombinant, Produced, Transfection, Control, Activity Assay

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 5. γ-carboxylated Gas6 reduces the tagged Mertk construct on cell membranes of THP-1 cells. (A) THP-1s expressing the tagged construct were starved for 18 h in serum-free RPMI and treated for 3 h. Flow cytometry data shows positive GFPs without staining. As expected, CHX treatment after 3 h reduces the expression of the construct. Gas6-VK, used at a concentration > 10 nM, decreases the FLAG-PE signal more when compared to other treatments known to induce cleavage (PMA, LPS). GW280264X is used as a control for Mertk cleavage. (B) Histogram analysis of “A.” Gas6-VK induces a shift in the FLAG-PE signal, showing that less surface Mertk is present. γ-Carboxylated Gas6 induced the degradation of Mertk on the cell membrane. (C) THP-1s treated with γ-Carboxylated Gas6 at a 10 nM concentration show increased MerTK phosphorylation, detected by immunoblotting against pMerTK. (D) THP-1s expressing the tagged construct, treated with >10 nM of Gas6-VK + 1 µM of PS, show a reduced level of both domains of the tagged construct, suggesting that the Gas6-VK + PS treatment is leading to a degradation of the full-length receptor.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Construct, Expressing, Flow Cytometry, Staining, Concentration Assay, Control, Membrane, Phospho-proteomics, Western Blot

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 6. Confocal imaging of the GFP-tagged MerTK construct displayed the differential localization of MerTK upon ligand stimulation and the inhibition of proteases. (A) DAPT and MG132 treatments induce increased cytoplasmic GFP signals compared to untreated cells. (B) Confocal imaging shows GFP localized on the cell membrane, indicating the presence of tagged Mertk constructs on the cell surface. γ-Carboxylated Gas6-treated tagged THP-1 cells showed a reduction in the MerTK construct from the membrane and the localization in lysosomes. (C) Quantification of the GFP fluorescence intensity per cell, calculated from confocal images in mock, γ-carboxylated Gas6, and non-γ-carboxylated Gas6, with the bar plots showing the mean and standard error of each treatment. ns; non-significant; *** p < 0.001; **** p < 0.0001.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Imaging, Construct, Inhibition, Membrane, Fluorescence

Journal: International journal of molecular sciences

Article Title: Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

doi: 10.3390/ijms25084404

Figure Lengend Snippet: Figure 7. γ-carboxylation of Gas6 induces Mertk degradation independent of phosphorylation. (A) Mutants of the tagged construct were created. K619M, a substitution in the ATP-binding site (underlined) of the Mertk kinase domain, inhibits autophosphorylation by preventing ATP binding and the exchange of phosphate molecules. * Amino acid position for ADAM17 cleavage. (B) Constructs are treated with 10 nM ofGas6-VK for 30 min after a 6 h serum starvation. With the ability to bind ATP and phosphorylate, the WT construct becomes phosphorylated while the kinase dead K619M mutant does not. (C) Mutant constructs were treated with 3 µM of GW280264X (an ADAM17 inhibitor; GW), 5 µM of DAPT (a γ-secretase inhibitor), or 10 µM of MG132 (a protea- somal inhibitor) for 4 h. Results show that the absence of the K619M C-terminal fragment from the MG132 treatment with the addition of GW indicates the fragment is a product of cleavage. (D) Confocal images indicated that the K619M mutants have more cytoplasmatic c-Mertk than WT Mertk with MG132 treatment. (E) Comparison of the contrasting pathways between Notch Recep- tor and MerTK Signaling Models. In the absence of a ligand, Notch is not cleaved, while MerTK undergoes homeostatic cleavage, leading to proteasomal degradation. Upon ligand binding, Notch is cleaved at the ADAM 17 site, revealing the gamma-secretase site. Subsequent gamma-secretase cleavage releases the Notch intracellular domain, translocating it to the nucleus for transcriptional activation. Conversely, MerTK, upon ligand (Gas6) interaction, is internalized into endosomal compartments and localizes within lysosomes.

Article Snippet: Concentrated Gas6 reagents were analyzed for concentration via Western blotting using a Gas6 standard (R&D Systems, 885-GSB-050).

Techniques: Phospho-proteomics, Construct, Binding Assay, Mutagenesis, Comparison, Ligand Binding Assay, Activation Assay

Journal: 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: 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: EMBO Reports

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

doi: 10.15252/embr.202050078

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

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

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

Journal: EMBO Reports

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

doi: 10.15252/embr.202050078

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

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

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

Journal: EMBO Reports

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

doi: 10.15252/embr.202050078

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

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

Techniques: Expressing

Journal: ASN NEURO

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

doi: 10.1177/1759091416668430

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

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

Techniques: Expressing, Gene Expression, Control, Western Blot

Journal: ASN NEURO

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

doi: 10.1177/1759091416668430

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

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

Techniques: Expressing

Journal: ASN NEURO

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

doi: 10.1177/1759091416668430

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

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

Techniques: Cell Culture, Fluorescence, Control

Journal: ASN NEURO

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

doi: 10.1177/1759091416668430

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

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

Techniques: Staining, Comparison

Journal: ASN NEURO

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

doi: 10.1177/1759091416668430

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

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

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

Journal: ASN NEURO

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

doi: 10.1177/1759091416668430

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

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

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

Journal: ASN NEURO

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

doi: 10.1177/1759091416668430

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

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

Techniques: Cell Culture

Journal: ASN NEURO

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

doi: 10.1177/1759091416668430

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

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

Techniques: Expressing, Cell Culture, Quantitative RT-PCR