Fig 1.
Structure and basic properties of DMF, MEF salts and Fumaric Acid.
DMF and MEF are esters of fumaric acid, which is not pharmacologically active. The active moiety(ies) of DMF and MEF confer their chemical and physical properties.
Fig 2.
DMF and MEF Differentially Modify KEAP1 Cysteine Residues.
KEAP1 transfected HEK 293FT cells were treated with DMF and MEF salts (Ca2+, Mg2+, Zn2+) at 3 μg/mL (A, C) or 6 μg/mL (B, D). Resulting cysteine modifications on KEAP1 were analyzed using mass spectrometry. Percent modification of KEAP1 cysteine residues with DMF or MEF was determined relative to DMSO control treated cells. (A, B) Representation of percent cysteine modification of analyzed KEAP1 cysteine residues in the presence of 3 (A) or 6 (B) μg/mL DMF or MEF. Each bar represents the means of quadruplicate determinations (± SD). (C, D) Box-whisker plots demonstrate the means, quartiles, and max-min of KEAP1 cysteine residues modified by greater than 10 percent in A and B. *, p<0.05. ***, p<0.001. ****, p<0.0001. P values are based on two-way analysis of variance (ANOVA) with Sidak’s post-test for multiple comparisons.
Fig 3.
NRF2 Translocation Induced by DMF and MEF Treatment.
Human astrocytes were treated with a mixture of MEF salts (Ca2+, Mg2+, Zn2+), DMF or the vehicle control DMSO. Extracts from harvested cells were separated into nuclear and cytoplasmic fractions and equal protein amounts from triplicate aliquots of each fraction were analyzed using an NRF2 TransAM DNA binding assay (A), and Western blots for NRF2, HDAC1 or β-actin (B). Red asterisks (*, p<0.01) indicate comparison of DMF versus MEF at indicated concentration. Black asterisks directly above bar plots (*, p<0.05, ***, p<0.001, ****, p<0.0001) refer to DMF and MEF versus DMSO control at indicated concentrations. P values are based on two-way ANOVA with Tukey’s post-test for multiple comparisons.
Fig 4.
DMF and MEF Produce Distinct Changes in Gene Expression in Astrocytes.
Human astrocytes were treated with a concentration-response of DMF, MEF salts (Ca2+, Mg2+, Zn2+) or fumaric acid for 24 hours, and analyzed for transcriptional changes in putative NRF2 target genes (NQO1, HMOX1, OSGIN1, TXNRD1, GCLC and SRXN1). Triplicate determinations (± SD) were normalized as a fold change relative to DMSO controls for each gene and probe set. *, p<0.01 for DMF versus MEF at indicated concentration. P values are based on two-way ANOVA with Tukey’s post-test for multiple comparisons.
Fig 5.
DMF and MEF Induce Different Effects on Cellular GSH.
Primary cultures of human astrocytes were incubated with 1 (A) or 3 (B) μg/mL DMF, MEF, or DMSO as a control. Treated cells were harvested after 0.0, 0.5, 1.0, 6, 12, and 24 hours of treatment, and total cellular GSH was measured as relative luminescence units (RLU). Each point represents the mean of triplicate determinations (± SD). Dotted line represents average basal RLU levels. *, p<0.01 for DMF versus MEF at indicated time point. P values are based on two-way ANOVA with Tukey’s post-test for multiple comparisons.
Fig 6.
DMF and MEF Induce Different Effects on Extracellular GSH.
Primary cultures of human astrocytes were incubated with 1 (A) or 3 (B) μg/mL DMF, MEF or DMSO as a control. Media was collected from treated cells after 0.0, 0.5, 1.0, 6, 12 or 24 hours of treatment, and total extracellular glutathione was measured as relative luminescence units (RLU). Each point represents the mean of triplicate determinations (± SD). Dotted line represents average basal RLU levels. *, p<0.01 for DMF versus MEF at indicated time point. P values are based on two-way ANOVA with Tukey’s post-test for multiple comparisons.