Fig 1: AMPK phosphorylation is required for Ex-4-induced activation of the Gas6/Axl pathwayA, WB for p-AMPK and AMPK in BMDMs treated with MD, Ex-4 and Compound 5D. B, Densitometry analysis of p-AMPK. C, Molecular docking model of GLP-1R and AMPK. D, Co- IP for GLP-1R and AMPK. E-G, RMSD, Rg, and RMSF analyses of the GLP-1R–AMPK complex. H and I, Hydrogen bond counts and SASA of the complex. J and K, MM/GBSA energy contribution of key residues. L, FEL analysis of the complex. M, Chemical structure of BML-275. N, WB of Gas6 and p-Axl after BML-275 treatment. O and P, Densitometric quantification of Gas6 and p-Axl. Q, IF images of p-Axl (green) and Gas6 (pink) in BMDMs; scale bar = 100 μm. R, Quantification of fluorescence intensity. S, IF of IL-6 (green) and p21 (red) in BMDMs; scale bar = 100 μm. T, Quantification of IL-6 and p21. ∗, p < 0.05, ∗∗, p < 0.01, and ∗∗∗, p < 0.001.
Fig 2: Ex-4 attenuates macrophage senescence and efferocytosis dysfunction through activating Gas6 in SCI miceA, IF images of F4/80 (green), Gas6 (pink), F4/80 (blue), p-Axl (pink) at 7 dpi in spinal cords following AAV-shGas6 and Ex-4 treatment; scale bar = 200/100/50 μm. B, IF images of F4/80 (green), IL-6 (pink), F4/80 (blue), p21 (red); scale bar = 200/100/50 μm. C, IF images of F4/80 (green) and LAMP2 (pink); scale bar = 200/100/50 μm. D–H, Quantification of Gas6-, p-Axl-, IL-6-, p21-, and LAMP2-positive macrophage areas. ∗, p < 0.05, ∗∗, p < 0.01, and ∗∗∗, p < 0.001.
Fig 3: Ex-4 significantly activates Gas6 signaling in macrophagesA, Relative mRNA expression of Gas6 in BMDMs after Ex-4 treatment. B, WB results for Gas6 expression in BMDMs after Ex-4 treatment. C, Densitometry analysis of Gas6 expression. D, Representative IF labeling images of p-Axl (green) and Gas6 (pink) in BMDMs after Ex-4 treatment; scale bar = 50 μm. E, Quantitative fluorescence intensity analysis of p-Axl and Gas6. F, WB results for p-Axl expression in BMDMs after transfection with LV-shGas6. G, Densitometric analysis of p-Axl protein levels. ∗, p < 0.05, ∗∗, p < 0.01, and ∗∗∗, p < 0.001.
Fig 4: Gas6 is a critical downstream target of Ex-4 in alleviating macrophage senescence and efferocytosis impairmentA, SA-β-gal staining in BMDMs transfected with LV-shGas6; scale bar = 100 μm. B, Quantification of SA-β-gal-positive cells. C–G, Relative mRNA expression of SASP factors and senescence markers in LV-shGas6-transfected BMDMs. H, Live-cell imaging of BMDMs post-LV-shGas6 transfection; scale bar = 5 μm. I, IF images of NeuN (blue) and MAP2 (red) in neurons co-cultured with Gas6-deficient BMDMs; scale bar = 50/25 μm. J, Sholl analysis of neuronal branching. K, IF images of GFAP (green) and Aggrecan (red) in astrocytes co-cultured with Gas6-deficient BMDMs; scale bar = 100 μm ∗, p < 0.05, ∗∗, p < 0.01, and ∗∗∗, p < 0.001.
Fig 5: Gas6 knockdown impairs EX-4's effects on macrophage senescence and efferocytosis dysfunctionA, Representative IF images of IL-6 (green) and p21 (red) in BMDMs after transfection with LV-shGas6; scale bar = 100 μm. B, Quantitative analysis of IL-6 and p21 fluorescence intensity. C, Representative IF labeling images of CFSE-labeled apoptotic neurons (green) and LAMP2 (pink) in BMDMs after transfection with LV-shGas6; scale bar = 50 μm. D, Quantitative fluorescence intensity of CFSE and LAMP2. E, Quantification of apoptotic neuron engulfment by BMDMs within 80 min under different conditions. F, FCM plots of neuronal apoptosis. G, Quantification of apoptotic neurons. H, Quantitative analysis of GFAP and Aggrecan fluorescence in the co-culture system. ∗, p < 0.05, ∗∗, p < 0.01, and ∗∗∗, p < 0.001.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Mouse Gas6 Protein, CF