Fig. 1.

GT-induced autophagy in mouse primary cortical astrocytes. (A) Representative immunoblot of LC3-II/LC3-I expression in astrocytes treated with GT for 24 h. (B) Immunoblot study of LC3-II/LC3-I expression in astrocyte treatment with 10 μg/mL GT for several time points. Densitometry analysis of LC3-II expressions was performed using Image J. All statistical data are indicated as mean ± SEM (n = 3,*P < 0.05). (C) For immunocytochemistry analysis, astrocytes were treated with 10 μg/mL of GT for 24 h. Astrocytes were fixed and stained with Alexa fluor 555–conjugated anti-GFAP mouse mAb (red) and Alexa fluor 488–conjugated anti-LC3 antibody (green). Representative images of the cells were taken with a confocal microscope, and arrows indicate LC3 puncta. The numbers of LC3 puncta were quantified in at least 3 independent random areas of each slide, and at least 5 cells were counted (**P < 0.01). (D) GFAP intensities were determined via statistical analysis from confocal microscopic data counted in each individual experiment (n = 3, ns = not significant). (E) Astrocytes were pretreated (30 min) with Ki16425 or LPA 18:1 (used as a positive control) and treated with 10 μg/mL of GT for 24 h. LC3-II/LC3-I expression was determined by Western blotting. Statistical analysis was performed by two-way ANOVA, and data are indicated as mean ± SEM (n = 3, **P < 0.01, ns = non-significant).

ANOVA, analysis of variance; DAPI, 4′,6-diamidino-2-phenylindole; GFAP, Glial fibrillary acidic protein; GT, gintonin; LPA, lysophosphatidic acid; mAb, monoclonal antibody; SEM, standard error of mean.