Journal: bioRxiv

Article Title: EndoNB: A general strategy to study the internalization of cell surface proteins

doi: 10.1101/2025.06.08.658482

Figure Lengend Snippet: a , Structural alignment of the hybrid and β-I domains of wild-type and Alfa-tagged Integrin β1. b , Schematics showing the effect of 12G10 and Mab13 antibodies on integrin β1 activation state. These antibodies act by stabilizing the integrins in their active or inactive state. c , Quantification of integrin β1 uptake by flow cytometry using EndoNB in the presence of no antibodies (no treatment), or in the presence of 12G10 (activating), Mab13 (inactivating) or unrelated (anti integrin αvβ3 antibody). (n = 12, each n represents the median fluorescence of 2000-8000 cells). d , Structural alignment of the extracellular region of wild-type and Alfa-tagged transferrin receptor (TFR). Wild type: PDB 1suv, while Alfa-tagged was generated by AlphaFold 3. See for comparison of Alfa-tagged dimeric and monomeric TFR. e , Schematics showing the experimental design to test if transferrin receptor uptake is stimulated by the presence of transferrin. f , Quantification of transferrin receptor uptake for 15 or 30 minutes by flow cytometry using EndoNB in the presence of increasing concentrations of transferrin. (n = 4, each n represents the median fluorescence of 2000-8000 cells). See for quantification of transferrin-AlexaFluor488 signals at each condition. g , Structural alignment of the transmembrane and extracellular region of wild-type and Alfa-tagged AXL. Both structures generated with AlphaFold 3. h , Quantification of AXL uptake by flow cytometry using EndoNB in the absence or presence of the AXL ligand GAS6 (1ug/ml). (n = 4, each n represents the median fluorescence of 2000-8000 cells). See for comparison with the conditions with and without 3C. ns = non-significant, * p> 0.05, **** p> 0.0001. ANOVA with Tukey’s post hoc analysis.

Article Snippet: SNAP-JF646 substrate was a kind gift by Luke Lavis via the open chemistry team (jJanelia); Transferrin-Alexa Fluor 488 (Thermo Fisher scientific, #T13342); Phalloidin-iFluor 488 (AAT Bioquest, #23115); GAS6 recombinant protein (MedChemExpress via Nordic Biosite HY-P700724-20).

Techniques: Activation Assay, Flow Cytometry, Fluorescence, Generated, Comparison

Journal: bioRxiv

Article Title: EndoNB: A general strategy to study the internalization of cell surface proteins

doi: 10.1101/2025.06.08.658482

Figure Lengend Snippet: a , Structural alignment of the monomeric and dimeric extracellular region of wild-type and Alfa-tagged transferrin receptor (TFR, PDB). Wild type: PDB 1suv, while Alfa-tagged was generated by AlphaFold 3. b , Quantification of transferrin (AlexaFluor488) in each condition measured in . c , Quantification of AXL uptake by flow cytometry using EndoNB in the absence or presence of the AXL ligand GAS6 (1µg/ml) and also in the presence or not of 3C. (n = 4, each n represents the median fluorescence of 2000-8000 cells). ns = non-significant, ** p> 0.01, *** p> 0.001, **** p> 0.0001. ANOVA with Tukey’s post hoc analysis.

Article Snippet: SNAP-JF646 substrate was a kind gift by Luke Lavis via the open chemistry team (jJanelia); Transferrin-Alexa Fluor 488 (Thermo Fisher scientific, #T13342); Phalloidin-iFluor 488 (AAT Bioquest, #23115); GAS6 recombinant protein (MedChemExpress via Nordic Biosite HY-P700724-20).

Techniques: Generated, Flow Cytometry, 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 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: Journal of Inflammation (London, England)

Article Title: The PPAR-γ antagonist GW9662 elicits differentiation of M2c-like cells and upregulation of the MerTK/Gas6 axis: a key role for PPAR-γ in human macrophage polarization

doi: 10.1186/s12950-015-0081-4

Figure Lengend Snippet: GW9662 induces M2c-like cells that upregulate MerTK and its ligand Gas6. (A-C) Healthy monocytes were cultured in serum-free medium in the absence of cytokines or growth factors (M0 differentiation), with or without the PPAR-γ antagonist GW9662 (2.5-10 μM), for 4 days; when specified, the PPAR-γ agonist rosiglitazone (1 μM) was added. Expression of MerTK, CD163 and CD16 was measured by flow cytometry. (D-E) Gas6 production levels were quantified by ELISA in culture medium, upon incubation with or without GW9662 (2.5-10 μM) of otherwise untreated cells (M0 conditions), LPS (50 ng/ml; M1 conditions) or IL-4 (20 ng/ml; M2a conditions) exposed cells. (A-E) Pooled data are represented as mean values ± SEM. Analysis was performed using one-way repeated measures ANOVA with Newman-Keuls multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. When not specified by additional graphic signs, statistical annotations (asterisks) refer to comparisons with respect to the relative GW9662 untreated control group. Each set of data is representative of three independent experiments.

Article Snippet: Biotinylated goat polyclonal anti-human Gas6 antibody (R&D Systems), followed by HRP-conjugated streptavidin (Biolegend), was used for detection.

Techniques: Cell Culture, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Incubation, Control

Journal: Mediators of inflammation

Article Title: Growth Arrest-Specific 6 Enhances the Suppressive Function of CD4 + CD25 + Regulatory T Cells Mainly through Axl Receptor.

doi: 10.1155/2017/6848430

Figure Lengend Snippet: Figure 6: Gas6 modulates the Foxp3 and CTLA4 expression mainly through Axl receptor. (a–d) CD4+CD25+Tregs were treated with anti- Axl or anti-Mertk Abs or PBS in the presence of 100 ng/ml rmGas6. After 24 h of incubation, the expression of CTLA-4 and Foxp3 was determined by flow cytometry (𝑛= 4/group). ∗𝑃< 0.05 compared with the value for the Gas6 group, and #𝑃< 0.05 compared with the value for rmGas6+anti-Mertk group. (e–h) The expression of CTLA-4 and Foxp3 in Tregs with or without Axl knockout was determined by flow cytometry. ∗𝑃< 0.05 compared with the value for the Gas6 group.

Article Snippet: To investigate the effect of Gas6 on CD4+CD25+Tregs in vivo, healthy mice were administered 1, 3, or 6 μg/mouse of rmGas6 (8310-GS, R&D Systems, Minneapolis, MN) via tail vein.

Techniques: Expressing, Incubation, Flow Cytometry, Knock-Out

Journal: Molecular Cancer Therapeutics

Article Title: Evaluation of the Role of AXL in Fusion-positive Pediatric Rhabdomyosarcoma Identifies the Small-molecule Inhibitor Bemcentinib (BGB324) as Potent Chemosensitizer

doi: 10.1158/1535-7163.mct-23-0285

Figure Lengend Snippet: Figure 2. Gas6 stimulates Axl signaling in FP-RMS cells. A, Representative Western blot images showing AXL expression in IC-pPDX-104 and Rh41 WT, KO (sgAXL-1) and OE lines. GAPDH was used as loading control. B, Morphologic appearance of IC-pPDX-104 and Rh41 cells with AXL WT, KO and OE. Scale bar, 70 mm. C, Schematic representation of the AXL signaling pathway. D, Representative Western blot images of AXL WT, KO (sgAXL-1) and OE cells stimulated with 400 ng/mL GAS6 for 10 minutes. E, Quantification of Western blots measuring the phosphorylation of downstream targets of AXL in AXL WT, KO (sgAXL-1) and OE cells stimulated with 400 ng/mL GAS6 for 10 minutes. Data are represented as mean SEM of the indicated number of independent biological replicates; ordinary two-way ANOVA with Sidak multiple comparisons test. ns, nonsignificant.

Article Snippet: The next day, we stimulated cells with the indicated concentration of GAS6 (R&D Systems, 885-GSB-050) for 10 minutes at 37 C, and processed them for Western blot analysis.

Techniques: Western Blot, Expressing, Control, Phospho-proteomics

Journal: Molecular Cancer Therapeutics

Article Title: Evaluation of the Role of AXL in Fusion-positive Pediatric Rhabdomyosarcoma Identifies the Small-molecule Inhibitor Bemcentinib (BGB324) as Potent Chemosensitizer

doi: 10.1158/1535-7163.mct-23-0285

Figure Lengend Snippet: Figure 5. Axl contributes to the migratory phenotype of Rh41 cells. A–C, Effect of AXL expression levels on the migratory phenotype of Rh41 cells. Representative images of Rh41 AXL WT (sgAAVS1), KO (sgAXL-1, sgAXL-2) and OE cells (A) and quantification (B) of wound-healing assay. Scale bar, 100 mm. C, Wound area remaining after 20 hours. D–F, Effect of bemcentinib on the migratory phenotype of Rh41 cells. Representative images (D) and quantification (E) of wound-healing assay performed on Rh41 WT cells treated with the indicated concentrations of bemcentinib. Scale bar, 100 mm. F, Wound area remaining after 12 hours in Rh41 cells. G and H, Effect of bemcentinib and AXL stimulation with GAS6 on the migratory phenotype of AXL KO cells. G, Quantification of wound-healing assay performed on Rh41 AXL WT (sgAAVS1) and KO (sgAXL-1, sgAXL-2) cells treated with the indicated concentrations of bemcentinib or with 400 ng/mL GAS6. H, Wound area remaining after 15 hours in Rh41 cells. Data are represented as mean þ SEM of n ¼ 3 biological replicates. Ordinary one-wayANOVA with Dunnett multiple comparison test againstthe control group (WT or 0 mmol/L bemcentinib).

Article Snippet: The next day, we stimulated cells with the indicated concentration of GAS6 (R&D Systems, 885-GSB-050) for 10 minutes at 37 C, and processed them for Western blot analysis.

Techniques: Expressing, Wound Healing Assay, Comparison, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Gas6 delays senescence in vascular smooth muscle cells through the PI3K/ Akt/FoxO signaling pathway.

doi: 10.1159/000373940

Figure Lengend Snippet: Fig. 2. Gas6 delays the senescence process in VSMCs. (A and B) Western blots demonstrating that cells trea- ȋʹͷͲȀȌ͵ϐ ͳ Ͷ and p21Cip1 expression. (C) The ef- fects of Gas6 on the IS and RS models were examined using western blotting. In the IS model, the Gas6-tre- ated cells showed low p21Cip1 and p16 Ͷ expression. In the RS model, the Gas6-treated cells also showed low p21Cip1 and p16 ͶǤȋȌǦȾǦ Ǧ ϐ Ǧ Ǧ RS models. (E and F) When these cells were treated with Axl-Fc, the levels of p16 Ͷ and p21Cip1 and the ǦȾǦ ϐ Ǥ (n=3 in each case). The values are presented as the mean±SD. *P<0.05, **P<0.01 and ***P<0.001 compared with the corresponding blank group; #P<0.05, ##P<0.01 and ###P<0.001 compared with the corresponding blank group. Bar, 200 μm.

Article Snippet: Recombinant mouse Gas6 (Gas6) protein and the recombinant mouse Axl-Fc protein fragment (Axl-Fc) were purchased from R&D Systems (St. Paul, MN, USA).

Techniques: Western Blot, Expressing

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Gas6 delays senescence in vascular smooth muscle cells through the PI3K/ Akt/FoxO signaling pathway.

doi: 10.1159/000373940

Figure Lengend Snippet: Fig. 3. Axl is the primary receptor in the Gas6-mediated anti-senescence effect. (A) Western blotting and SA- ȾǦͳ Ͷ and p21Cip1ϐ in R428-treated cells regardless of Gas6 treatment compared with the two R428-free cell groups, whereas ϐ ͶʹͺǦ ǤȋȌǦȾǦ ͶʹͺǦ ϐ compared with the two R428-free cell groups. All the results shown are from representative experiments (n=3 in each case). The values are presented as the mean±SD. **P<0.01 and ***P<0.001 compared with the non-R428-treated group; ##P<0.01 and ###P<0.001 compared with the non-R428-treated group. Bar, 200 μm.

Article Snippet: Recombinant mouse Gas6 (Gas6) protein and the recombinant mouse Axl-Fc protein fragment (Axl-Fc) were purchased from R&D Systems (St. Paul, MN, USA).

Techniques: Western Blot

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Gas6 delays senescence in vascular smooth muscle cells through the PI3K/ Akt/FoxO signaling pathway.

doi: 10.1159/000373940

Figure Lengend Snippet: Fig. 4. Gas6 promotes the transition from G1 to S phase. Cell cycle analyses showing that Gas6-treated cells in both the IS and the RS models showed higher percentages of S phase and a lower percentage of G1 phase Ǧ Ǧ Ǣȋ Ȍϐ with the corresponding controls. (B) EdU staining results showed that the positive staining rates in both the IS and the RS models after Gas6 treatment were higher than in controls. All the results shown are from representative experiments (n=3 in each case). The values are presented as the mean±SD. *P<0.05 compared with the non-Gas6-treated group; #P<0.05 compared with the non-Gas6-treated group.

Article Snippet: Recombinant mouse Gas6 (Gas6) protein and the recombinant mouse Axl-Fc protein fragment (Axl-Fc) were purchased from R&D Systems (St. Paul, MN, USA).

Techniques: Staining

Journal: 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