Fig. 2

Metformin suppresses senescence of astrocytes in vitro. A Astrocytes were pretreated with metformin at indicated concentrations for 30 min and then stimulated with α-Syn PFF. Representative immunoblots of relative expression of p16 in astrocytes. B Quantification of relative expression of p16 in A (Three independent experiments). C–F qPCR measurement of the levels of IL-1α (C), IL-1β (D), MMP3 (E), and MMP9 (F) in astrocytes treated with metformin (0.2 mM) and α-Syn PFF (Six independent experiments). G, H Representative images of SA-β-gal staining and quantification of the percentage of SA-β-gal⁺ astrocytes over total astrocytes in astrocytes treated with metformin (0.2 mM) and α-Syn PFF (Three independent experiments). I Astrocytes were pretreated with metformin (0.2 mM) for 30 min and then stimulated with MPP⁺. Representative immunoblots of relative expression of p16 in astrocytes. J Quantification of relative expression of p16 in I (Three independent experiments). K, L Representative images of SA-β-gal staining and quantification of the percentage of SA-β-gal⁺ astrocytes over total astrocytes in astrocytes treated with metformin (0.2 mM) and MPP⁺ (Three independent experiments). M Astrocytes were cultured for 7 days or 40 days with or without metformin (0.2 mM). Heatmap of relative mRNA levels of SASP as indicated in astrocytes. N–P qPCR measurement of IL-6 (N), MMP3 (O), and p16 (P) mRNA expression in astrocytes (Three independent experiments). Q, R Representative immunoblots and quantification of relative expression of p16 in astrocytes (Three independent experiments). S, T Representative images of SA-β-gal staining and quantification of the percentage of SA-β-gal⁺ astrocytes over total astrocytes in astrocytes (Three independent experiments). U, V Immunofluorescence and quantification of lamin B1 in GFAP⁺ astrocytes (Three independent experiments). DAPI stains nucleus (blue). The data shown are the mean ± SEM. One-way ANOVA with Tukey’s post-hoc tests were used. *p < 0.05, **p < 0.01, ***p < 0.001