TYRO3 Antibody - #DF2680

Fig. 6. AXL receptor tyrosine kinase (AXL)/phosphatidylinositol 3-kinase (PI3K)/AKT serine/threonine kinase (AKT) pathway mediates the anti-ferroptotic effects of growth arrest-specific 6 (Gas6) and vitamin K. (A) Evaluation of anti-ferroptotic effect of Gas6 in chondrocytes. Chondrocytes were pretreated with indicated Gas6 concentrations for 2 h followed by treated with RSL3 (0.5 μM) for 24 h. (B) The protein expression levels of TAM receptors (AXL, TYRO3) in chondrocytes were detected by Western blot analysis. Chondrocytes were treated with RSL3 (0.5 μM, 24 h), vitamin K1(VK1, 10 μM, 24 h) or Gas6 (100 ng/mL, 2 h). (C) Cell viability was detected by Cell Counting Kit-8 (CCK-8). Chondrocytes were transfected with AXL small interfering RNA (si-AXL, #1, #3), TYRO3 small interfering RNA (si-TYRO3, #2, #3) or negative control siRNA (si-NC). A total of 24 h after transfection, chondrocytes were pretreated with Gas6 (100 ng/mL) for 2 h followed by treatment with RSL3 (0.5 μM) for 24 h. (D, E) Intracellular malondialdehyde (MDA) analysis (D) and reduced glutathione/oxidized glutathione (GSH/GSSG) (E) ratio of chondrocytes. Chondrocytes were transfected with si-AXL #1 or si-NC for 24 h. A total of 24 h after transfection, chondrocytes were pretreated with Gas6 (100 ng/mL) for 2 h followed by treatment with RSL3 (0.5 μM) for 24 h. (F) Intracellular ferrous levels detected by FerroOrange probe and lipid peroxidation evaluated by BODIPY 581/591 C11 staining of chondrocytes (green: oxidized lipids; red: lipids; and blue: Hoechst). (G) The mRNA levels of Mmp13, Adamts4, Col2a1 and Acan in chondrocytes were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). (H) The protein expression levels of matrix metalloproteinase 13 (MMP13), ADAMTS4, collagen type II (COL II) and Aggrecan in chondrocytes were detected by Western blot analysis. (I) The phosphorylation of AXL (P-AXL) was detected by Western blot analysis. After pretreated with R428 (AXL inhibitor, 5 μM) for 15 min, chondrocytes were treated with Gas6 (100 ng/mL) for 2 h followed by treatment with RSL3 (0.5 μM) for 2 h. (J) Cell viability were detected by CCK-8. Chondrocytes were pretreated with R428 (5 μM) for 15 min followed by treated with Gas6 (100 ng/mL, 2 h) and RSL3 (0.5 μM, 24 h). (K) The P-AKT was detected by Western blot analysis. After pretreated with GDC-0941 (PI3K inhibitor, 5 μM) for 15 min, chondrocytes were treated with Gas6 (100 ng/mL) for 2 h followed by treatment with RSL3 (0.5 μM) for 2 h. (L) Cell viability were detected by CCK-8. Chondrocytes were pretreated with GDC-0941 (5 μM) for 15 min followed by treatment with Gas6 (100 ng/mL, 2 h) and RSL3 (0.5 μM, 24 h). (M) Cell viability was detected by CCK-8. Chondrocytes were transfected with si-AXL, si-TYRO3 or si-NC. A total of 24 h after transfection, chondrocytes were treated with VK1 (10 μM) and RSL3 (0.5 μM) for 24 h. (N) Cell viability were detected by CCK-8. Chondrocytes were pretreated with R428 (5 μM) or GDC-0941 (5 μM) for 15 min followed by treated with VK1 (10 μM) and RSL3 (0.5 μM) for 24 h. (O) Graphic abstract of our study indicating how vitamin K and Gas6 protects against osteoarthritis through inhibiting ferroptosis. Data are the mean ± standard deviation (SD) from three independent experiments with one-way analysis of variance (ANOVA) with Dunnett's post hoc test. ∗∗P < 0.01; ns: not significant. PBS: phosphate buffered saline;Ctrl: Control.