Fig. 3

4-EG inhibits Th1 and Th17 differentiation in vivo and in vitro. a Splenocytes harvested from vehicle- and 4-EG (100 mg/kg)-treated C57BL/6 EAE (n=8/group) at day 8 post-immunization were subjected to flow cytometry analysis to determine the frequency and number of IFNγ- and IL-17-expressing CD4⁺ T cells. b, c Naive splenocytes were polarized into Th1 and Th17 conditions in the presence or absence of 4-EG (200 μM). Forty-eight and 72 hours after incubation, cells were collected and subjected to flow cytometry analysis to assess the frequency of intracellular expression of IFNγ or IL-17 in CD4⁺ T cells. Data are representative of four independent experiments (n=2–3/technique replicates per experiment). Isotype controls (Iso) were used as a negative control to determine CD4⁺ cells positive for intracellular expression of IFNγ or IL-17. Statistical significance was determined as *p<0.05, **p<0.01, and ***p<0.001 by unpaired t test