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: Once confluence was achieved (usually 5 days post seeding), the cells underwent airlifting by removing the apical media entirely and replacing the basal media with SAEC ALI complete medium (450 ml Pneumacult TM ALI-S medium (STEMCELL, #05051) supplemented with 50 ml 10x ALI-S supplement (STEMCELL, #05052), 2.5 ml hydrocortisone, 5 ml maintenance system supplement (STEMCELL, #050502), 1 ml heparin (STEMCELL, #07980), and 5 ml penicilin-streptomycin) to promote cell differentiation.

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: Once confluence was achieved (usually 5 days post seeding), the cells underwent airlifting by removing the apical media entirely and replacing the basal media with SAEC ALI complete medium (450 ml Pneumacult TM ALI-S medium (STEMCELL, #05051) supplemented with 50 ml 10x ALI-S supplement (STEMCELL, #05052), 2.5 ml hydrocortisone, 5 ml maintenance system supplement (STEMCELL, #050502), 1 ml heparin (STEMCELL, #07980), and 5 ml penicilin-streptomycin) to promote cell differentiation.

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