Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) IPF signaling pathway was one of the increasingly upregulated pathways over time as revealed by IPA analysis. The gene set contributing to this pathway included distinct groups of early and late profibrotic associated genes (marked by black rectangles) that were serially and time dependently upregulated. Timepoint order forced, genes hierarchical clustered. (B- D) Fold change of mRNA expression levels of TAM RTK family, Axl , Mertk and Tyro3 over the course of repair. (E-F) Fold change of expression level of TAM RTK ligands, Gas6 and Pros1 over the course of repair. Data are shown as mean ± SEM of n= 3-7. P ≤ 0.05 (*); P ≤ 0.01 (**); P ≤ 0.001 (***); P ≤ 0.0001 (****) compared with stuffer control.

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) Schematic of the e xperimental setup to study AXL-GAS6/PROS1 interaction using aFRET in mouse lung tissue sections. Lungs were isolated on day 4 post DT. aFRET is a coincidence assay, labels both receptor (AXL) and ligands (GAS6 or PROS1) (see Methods for details). FRET occurs in the range of 1-10 nm. Scheme was created in BioRender. Geillinger-kästle, K. (2025) https://BioRender.com/h45x771 (B) Representative image of AXL-GAS6 interaction. (C) Representative image of AXL-PROS1 interaction. (B-C) Upper figures showed the mean of the donor lifetime (Ƭ) in the absence of acceptor. Lower figures showed the interaction state between AXL-GAS6/ PROS1 for each coincidental region (marked by yellow and black circles). Different coincidental regions possessed different interaction state. (D) Box and whiskers plots showed the heterogeneity of AXL-GAS6/PROS1 interaction states represented as FRET efficiency (Ef%). Ef% below 4% represents the Förster Radius (R 0 at 5.83nm) and not proteins, interactive state as marked by red dashed line. Data are shown as box and whiskers plot of n=5-6. Each dot represents median of Ef% for each mouse. P ≤ 0.05 (*). (E) Correlation plot between Ef% AXL-GAS6 and AXL expression. (F) Correlation plot between Ef% AXL-PROS1 and AXL expression. (G-H) Surface plasmon resonance response curve showing the binding of rhGAS6 and rhPROS1 in different concentration to rhAXL. (I-J) Concentration dependent binding of rhGAS6 and rhPROS1 to rhAXL. Different colored lines in (G-H) correspond to different colored dots in (I-J), which indicate different concentrations of rhGAS6 and rhPROS1. (K-L) GAS6 and PROS1 concentration in human BALF of non- diseased control, ILD/IPF-, and COPD patients. Data are shown as mean ± SEM of n= 4-8. P ≤ 0.01 (**); P ≤ 0.001 (***).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Isolation, Expressing, SPR Assay, Binding Assay, Concentration Assay, Control

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) 7664 is a tissue section from a healthy lung whereas 1015 and 1033 are sections from IPF patients with various advanced degrees of IPF. The panels show representative images mapping the interactive states of GAS6 and PROS1 with AXL (pseudo-colour heat map of (Ef%) on the expression level of GAS6 and PROS1 (fluorescence images in grey scale). The individual violin plots quantify the heterogeneity of interactive states for both ligands and the receptor. The dotted line at 4% Ef represents the Förster Radius (R 0 ). Values below 4% are not representative of ligand-receptor interactions. (B) Global violin plots of all coincident regions (per pixel) of AXL-GAS6 and AXL-PROS1. Each global violin plot represents 2x10 6 data points. To determine the p values for AXL-GAS6 and AXL-PROS1, using non-parametric Mann- Whitney U test, we utilised 1000 randomly generated data points of the 2x10 6 . Healthy lung has similar distribution of AXL-GAS6 and AXL-PROS1 interactions, whereas the two patient samples show, in one case (1015) no interaction of AXL-GAS6 or AXL-PROS1 and another case (1033) a significantly higher distribution of AXL-PROS1 versus AXL-GAS6. The p values between AXL-GAS6 and AXL-PROS1 are 1.9x10 -3 (7664), 2.4x10 -1 (1015) and 8.2x10 - respectively. The dotted line at 4% Ef represents the Förster Radius (R 0 ).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Expressing, Fluorescence, MANN-WHITNEY, Generated

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: AXL is predominantly expressed in basal and aberrant basaloid cells, promoting proliferation via AXL-GAS6 signaling. (A) UMAP plots visualize AXL and its ligands, GAS6 and PROS1 normalized expression in the epithelial cell compartment of an integrated scRNA-seq IPF atlas. (B) Quantification of AXL and its ligands expression in epithelial cells of IPF patients and the non-diseased controls. (C) Expression of AXL and its ligands within the epithelial cells compartment in the lung of PF patients and the non-diseased controls. (D-E) AXL and its ligands expression in SAEC under submerged culture condition. (F) Representative western blot image validating knockout of AXL via CRISPR/Cas9 in 2 different donors. AXL (140 KDa, red bands), loading control ß-Actin (42 KDa, green bands), and M is protein marker. (G) Quantification of AXL expressions of each donor before and after CRISPR/Cas9 mediated knockdown. Experiments were done in duplicate. (H) Proliferation of AXL WT and AXL KD SAEC as assessed by BrdU in all donors 48 hours post seeding. (I) Correlation between AXL expression in (G) and SAEC proliferation in (H). (J) Proliferation of AXL WT and (K) AXL KD SAEC as assessed by BrdU 48 hours post treatment with rhGAS6/ rhPROS1/ combination of both proteins. WT: Wildtype; KD: Knockdown. Data are shown as mean ± SEM of n= 4 – 7. P > 0.05 (ns/ non-significant); P ≤ 0.05 (*); P ≤ 0.001 (***).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Expressing, Western Blot, Knock-Out, CRISPR, Control, Marker, Knockdown

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: (A) SAEC basal cells were allowed to differentiate for 23 days. On day 23 post ALI, 5 ng/ml rhTGF-ß was added to the culture. On day 26 post ALI, rhTGF-ß was added together with rhGAS6, rhPROS1, or a combination of both. Analysis was performed on day 29 post ALI. Scheme was created in BioRender. Geillinger-kästle, K. (2025) https://BioRender.com/k46g355 . (B) GAS6 and (C) PROS1 concentration in the SAEC cell culture supernatant post rhTGF-ß treatment compared to the BSA control. (D) Epithelial barrier integrity as measured by FITC-dextran permeability assays. (E) Fold change of AXL mRNA expression as measured by qPCR. (F) Correlation between slope measured by FITC- dextran permeability assay (D) and ΔCT of AXL (r= -0.9519 R 2 = 0.9062) measured by qPCR (E). Data are shown as mean ± SEM of n= 4 – 5. P > 0.05 (ns/ non-significant); P ≤ 0.05 (*); P ≤ 0.01 (**).

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Concentration Assay, Cell Culture, Control, Permeability, Expressing, FITC-Dextran Permeability Assay

Journal: bioRxiv

Article Title: AXL-GAS6/PROS1 Interaction: A Critical Switch Between Aberrant- and Healthy Repair Following Alveolar Lung Injury

doi: 10.1101/2025.04.07.647567

Figure Lengend Snippet: After injury, the normal repair process involves an initial inflammatory response, followed by AXL- GAS6 interaction, which promotes cell proliferation. Once sufficient proliferation is achieved, AXL-PROS1 binding occurs, halting proliferation and triggering cell differentiation. This balance between proliferation and differentiation ensures proper repair, leading to healthy alveoli. However, dysbalanced AXL signaling due to repeated injury and persistently high TGF- ß levels, can result in aberrant repair and irreversible fibrosis (IPF). This may occur due to high-affinity interaction between AXL and GAS6 prevents PROS1 from effectively binding to AXL. As a result, excessive proliferation occurs with insufficient differentiation, leading to aberrant repair and fibrosis. Scheme was created in BioRender. Geillinger-kästle, K. (2025) https://BioRender.com/q77c168.xss

Article Snippet: GAS6 was measured in human BALF or SAEC supernatant using the human GAS6 DuoSet ELISA kit (R&D Systems, #DY885B) and the human PROS1 ELISA Kit (Aviva Systems Biology, #OKBB010207).

Techniques: Binding Assay, Cell Differentiation