Fig. 3

Metformin normalizes mitochondrial function in senescent astrocytes by Mfn2. A Astrocytes were pretreated with metformin (0.2 mM) for 30 min and then stimulated with MPP⁺. Representative images of JC-1 in astrocytes were analyzed using confocal microscopy (Three independent experiments). DAPI stains nucleus (blue). B, C Representative images and quantification of depolarized mitochondria analyzed by flow cytometry (Five independent experiments). D, E Representative images and quantification of mitochondrial ROS level analyzed by flow cytometry (Six independent experiments). F, G Representative images and quantification of mitochondrial ROS immunofluorescence intensity in immunofluorescence using ImageJ software (Six independent experiments). DAPI stains nucleus (blue). H–J qPCR measurement of mtDNA in cytosol of astrocytes pretreated with metformin (0.2 mM) and then stimulated with MPP⁺ (H), α-Syn PFF (I) or cultured for 40 days (J) (Three independent experiments). K, L Representative immunoblots and quantification of relative expression of Mfn2 in astrocytes treated with metformin (0.2 mM) and MPP⁺ (Three independent experiments). M, N Astrocytes were transfected with empty vector (pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO) or Mfn2 plasmid for 48 h and then treated with metformin (0.2 mM) and MPP⁺. Representative images and quantification of mitochondrial ROS level analyzed by flow cytometry (Five independent experiments). O, P Representative images and quantification of dysfunctional mitochondria analyzed by flow cytometry (Four independent experiments). Q–S qPCR measurement of mtDNA in cytosol of astrocytes stimulated with MPP⁺ (Q), α-Syn PFF (R) or cultured for 40 days (S) (Three independent experiments). The data shown are the mean ± SEM. One-way ANOVA with Tukey’s post-hoc tests were used (C, E, F, H–J, L). Two-way ANOVA with Tukey’s post-hoc tests were used (N, O, Q–S). **p < 0.01, ***p < 0.001, NS: no significant