Fig. 6

ATO induces CD4⁺ T cell apoptosis through the mitochondrial pathway. CD4⁺ T cells were sorted from the spleen of normal mice and cultured with Con A or ATO (0, 1, and 2 μM) for 24 h. a, c Annexin V/PI was performed to assess CD4⁺ T cell apoptosis (n = 3). b, d The mitochondrial membrane potential was monitored by loading with JC-1 probe. J-aggregates indicates high membrane potential, and J-monomers indicates low membrane potential (n = 3). e Mitochondrial ultrastructure as observed by TEM. Boxes denote magnified areas. f The protein level of Bax, Bcl-2, cleaved-Caspase 9, and cleaved-PARP was analyzed by western blot. h On day 22 post-immunization, CD4⁺ T cells isolated from the spleen were used to extract total protein. The protein levels of Bax, Bcl-2, cleaved-Caspase 9, and cleaved-PARP in CD4⁺ T cells were detected by western blot. g, i The activity of Caspase 3 was determined with the Caspase 3 Activity Assay kit (n = 3). Data are representative of three independent experiments. *p < 0.05, ***p < 0.001, and ****p < 0.0001 vs. the activated or control group; #p < 0.05 the EAE+ATO group vs. the EAE group