Fig. 1

Dimethyl fumarate does not appear to activate Nrf2 in time course experiments with human peripheral blood mononuclear cells (PBMCs). a PBMCs were left untreated (UNT), treated with vehicle (Veh), or treated with 200 µg/ml DMF for the time indicated. Nuclear extracts were isolated and 5 µg extract per well was used for TransAM Nrf2 ELISAs (Active Motif) performed in duplicate or triplicate. Positive (+) control Nrf2-transfected COS-7 nuclear extract was added to wells in the amounts indicated. Allogeneic donors for each time point: 30 min and 1 h, N = 4 each, 2 h N = 6, 4 h N = 5, and 6 h N = 3. *Indicates p < 0.05 compared to UNT. b PBMCs from a single donor were used for cAMP (left panel, Enzo Life Sciences) and Nrf2 ELISAs (right panel). For cAMP assay, cells were left untreated (UNT), treated with vehicle (Veh, ethanol), or treated with DMF at the indicated concentrations for 5 min; For Nrf2 assay, cells were treated with 200 µg/ml DMF for the time indicated. c PBMCs were left untreated (UNT) or treated with 200 µg/ml DMF for the time indicated, with the addition of FBS to 2.5% after the first 15 min of DMF treatment. Nuclear extracts were prepared using a commercially available kit (Cayman Chemical) and assayed for Nrf2 activation as above, performed in duplicate or triplicate. Five micrograms of positive control (+) Nrf2-transfected COS-7 nuclear extract was used. Allogeneic donors for each time point: 2 h UNT, 2 h DMF and 4 h DMF N = 6 each; 4 h UNT N = 3. *Indicates p < 0.05 compared to 2 h UNT