Fig. 2

4-EG suppresses encephalitogenic Th1 and Th17 infiltration of the CNS in EAE. At day 11–13 post-immunization, mononuclear cells were isolated from the brain and spinal cord of vehicle- and 4-EG (100 mg/kg)-treated C57BL/6 EAE mice (n=6/group), and the isolated mononuclear cells were then subjected to surface staining of CD4 followed by intracellular staining of IFNγ and IL-17. a–c The frequency determined by the percentages of CD4⁺ T cells in total acquired cells, and the percentages of IFNγ- or IL-17-expressing CD4⁺ T cells in total CD4⁺ T cells were analyzed in the brain and spinal cord of vehicle- and 4-EG-treated EAE mice by flow cytometry analysis. a–c The number of CD4⁺ T cells, CD4⁺ IFNγ⁺ T cells, and CD4⁺ IL-17⁺ T cells in the brain and spinal cord of vehicle- and 4-EG-treated EAE mice was also determined. One representative flow result of each condition is shown. Isotype controls (Iso) were used as a negative control to determine cells positive for the surface expression of CD4 a or CD4⁺ T cells positive for the intracellular expression of IFNγ or IL-17 b, c. Data are representative of three independent experiments (n=4–6/group per experiment). Statistical significance was determined as *p<0.05, **p<0.01, and ***p<0.001 by unpaired t test