Fig. 1

Dimethyl Fumarate (DMF) activates the Nrf2 pathway and inhibits the activation of proinflammatory markers in T98G glioma cells and primary astrocytes. A A schematic protocol for DMF treatment in the T98G glioma cell line. B Representative immunoblots for the lysates of T98G cells treated with or without DMF. DMF treatment activates the Nrf2 pathway in the T98G cell line. Cytosolic (Cyto) and nuclear (Nuc) fractions were subjected to immunoblotting using an anti-Nrf2 antibody. Fractionation was validated by immunoblotting for the fraction-specific marker proteins fibrillarin (nuclear marker) and SOD1 (cytosolic marker). Cytosolic and nuclear fractions were loaded with 10 µg of the protein per well. C Quantification of immunoblotting in B. Relative levels of nuclear Nrf2 normalized by the nuclear marker fibrillarin were quantified from three independent experiments and are expressed as means ± standard error of the mean (SEM) with p-values (n = 3, each group). *p < 0.05 (Student’s t-test). D Schematic protocol for DMF treatment in primary cultured A1 astrocytes. E Expression levels of mRNAs in DMF-treated primary astrocytes as determined by quantitative PCR. Relative expression levels for Nfe2l2 downstream genes, such as Hmox1 and Gclm; A1-astrocyte markers, such as H2d, H2t23, and Gbp2; and inflammatory and glial markers, such as C3, Socs3, and Gfap, are plotted as means ± SEM (Veh-Control: n = 3, DMF-Control: n = 3, Veh-A1 (TNFα/IL-1α/C1q): n = 3, and DMF-A1 (TNFα/IL-1α/C1q): n = 3). *p < 0.05 and **p < 0.01 (two-way ANOVA). F Expression levels of mRNAs in A1-induced primary astrocytes derived from WT and Nrf2^−/− mice determined by quantitative PCR. WT and Nrf2^−/− astrocytes were preincubated with TNFα/IL-1α/C1q in the presence or absence of DMF, as shown in D. Relative expression levels for Hmox1, H2-d, C3, and Socs3 are plotted as means ± SEM (n = 3, each group). *p < 0.05 and **p < 0.01 (two-way ANOVA). G Representative immunofluorescent images demonstrating the expression of C3 (Blue), SOCS3 (Red), and GFAP (Green) in Veh-treated control or A1 astrocytes (upper panels, G) and DMF-treated control or A1 astrocytes (lower panels, G). Scale bars: 20 µm. H Quantification of immunofluorescence images in G. Percentage of the area immunopositive for C3 and SOCS3 in Veh- or DMF-treated control and A1 astrocytes. Values are represented as means ± SEM (n = 3, each group). *p < 0.05 and **p < 0.01 (two-way ANOVA)