Figure 3.GAS alleviated CoCl2-induced autophagosome accumulation in HT22 cells. (A) HT22 was incubated with different concentrations of CoCl2 for 24 h, and cell viability was detected using the MTT assay (n=6). (B) Cells were incubated with different concentrations of GAS for 1 h and then treated with CoCl2 (200 μM) for 24 h. Cell viability was assessed via the MTT assay (n=6). (C) Representative Giemsa staining of HT22 cells (magnification, 200×; n = 3) (D) Immunoblots showing levels of LC3, p62, p-p62 (Thr349)in HT22 cells treated with various concentrations of CoCl2 (0, 100, 200, 400 and 800 μM) for 24 h. β-actin was used as the loading control. (E) HT22 cells were pretreated with GAS and BAPT-AM (0.5μM) for 1 h and then exposed to CoCl2 (200μM) for 24 h. Expression of LC3, p62, and p-p62 (Thr349) was detected by immunoblotting. β-actin was used as the loading control.
(F) Immunofluorescence analysis revealing the modulation of LC3 in HT22 cells with or without GAS for 24 h (n = 3). Autophagosomes were visualized (green puncta) using a Leica DMIRB at 800× magnification. In each independent experiment, 5 visual field cells were randomly selected and quantified, and are expressed as mean ± standard error of the mean (SEM). Immunofluorescence staining of p62 in HT22 cells. (G) Immunofluorescence analysis of p62 in HT22 cells, with or without GAS treatment for 24 h (n = 3). (H) Representative transmission electron microscopy images in HT22 cells. Arrows denote autophagosomes. Data are presented as the mean ± SEM. ##P< 0.01 versus control, *P< 0.05 versus CoCl2. GAS, gastrodin; CoCl2, cobalt chloride; LC3, microtubule-associated protein 1 light chain 3.
