Research Paper Volume 13, Issue 7 pp 9542—9565

Figure 5. GAS alleviated CoCl₂-induced intracellular Ca²⁺ abundance and CaMKII activation. (A) After HT22 cells were treated with CoCl₂ for different time points, the Ca²⁺ content was detected using a commercial calcium quantitative kit. (B) After HT22 cells were treated with different doses of CoCl₂ for 24 h, phosphorylated CaMKII α increases in a dose-dependent manner. β-actin was used as a loading control. (C) HT22 cells were pretreated with GAS and BAPT-AM (0.5 μM) for 1 h and then plated with CoCl₂ (200 μM) for 24 h; the Ca²⁺ content was detected using a commercial calcium quantitative kit. (D) HT22 cells were pretreated with GAS for 1 h and exposed to CoCl₂ (200 μM) for 24 h; cytosolic Ca²⁺ levels were measured by flow cytometry. (E) GAS, similar to calcium chelator (BAPTA-AM), can reduce the level of phosphorylated CaMKII. (F–G) Levels of CaMKII and phosphorylated CaMKII (Ser249) in HT22 cells treated with calcium chelator (BAPTA-AM) or calcium ionophore (A23187) were detected with or without GAS treatment (200 μM) for 24 h (n = 3). The experimental results were normalized to β-actin levels and are shown as fold changes relative to control cells. Data are presented as the mean ± standard error of the mean (SEM) from three independent experiments. ^##P< 0.01 versus control, ^*P< 0.05 versus CoCl₂. GAS, gastrodin; CoCl₂, cobalt chloride; CaMKII, Ca²⁺-calmodulin stimulated protein kinase II.