Fig. 6

4-EG suppresses GM-CSF expression in vivo and in vitro. a Splenocytes were harvested from vehicle- and 4-EG (100 mg/kg)-treated C57BL/6 EAE mice at day 8 post-immunization (n=5/group), and mononuclear cells were isolated from the brain and spinal cord of vehicle- and 4-EG-treated C57BL/6 EAE mice at day 11–13 post-immunization (n=6/group). Splenocytes and the isolated mononuclear cells were then subjected to surface staining of CD4 followed by intracellular staining of GM-CSF. The frequency and number of GM-CSF-expressing CD4⁺ T cells in the spleen, brain, and spinal cord were analyzed by flow cytometry analysis. Isotype controls (Iso) were used as a negative control to determine cells positive for the intracellular expression of GM-CSF. b Naive splenocytes were polarized into Th1 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 GM-CSF in CD4⁺ T cells. Iso were used as a negative control to determine CD4⁺ cells positive for the intracellular expression of GM-CSF. Data are representative of four independent experiments (n=2–3/technique replicates per experiment). Statistical significance was determined as *p<0.05, **p<0.01, and ***p<0.001 by Mann-Whitney U test