Dimethyl fumarate dmf

MIN6 cells at passage 30 were kindly gifted by Dr. Marcia Haigis (Harvard University, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 25 mmol/l glucose, supplemented with 15% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, 2 mmol/l L-glutamine, and 5 μL/l β-mercaptoethanol in humidified 5% CO₂, 95% air at 37°C as previously described [21 (link)]. The cells at passages 42–48 were used in the current study. There was no significant difference among the passages in their glucose responsiveness and cytotoxic susceptibility to a variety of stressors. H₂O₂ solution, glucose oxidase, S-nitroso-N-acetylpenicillamine (SNAP), sodium arsenite, tert-butylhydroquinone (tBHQ), β-mercaptoethanol, hexadimethrine bromide, dimethyl fumarate (DMF), and 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) were purchased from Sigma (Saint Louis, MO).

Dimethyl fumarate (DMF) (Sigma-Aldrich) was resuspended in 100% DMSO to 100mM and further diluted at indicated dilutions before use in all in-vitro experiments. For in-vivo experiments, DMF was dissolved in 100% DMSO or 0.8% methyl cellulose at 50mg/mL diluted at indicated dilutions before use.
Monomethyl fumarate (MMF), Diethyl fumarate (DEF), Dimethyl maleate (DMM), Diethyl maleate (DEM) and Fumaric acid (FA) were all obtained from (Sigma-Aldrich) and all resuspended in 100% DMSO to 100mM and further diluted at indicated dilutions before use in all in-vitro experiments.

Dimethyl fumarate (DMF) was provided by Sigma Aldrich Co. (St. Louis, MO, USA). PD98059 was provided by Calbiochem (La Jolla, CA, USA). Rabbit polyclonal antibody against phospho-p44/42 MAPK (p-ERK1/2, catalog n. 9101), p44/42 MAPK (ERK1/2, catalog n. 9102), phospho-AKT (catalog n. 4060S), AKT (catalog n. 9272S) and GAPDH (catalog n. 2118) were provided by Cell Signaling Technology (Danvers, MA, USA); Rabbit polyclonal antibody against Nrf2 (catalog n. ab137550) was provided by Abcam (Cambridge, UK). All reagents used for cell cultures and stripping buffer were purchased from Invitrogen Thermo Fisher Scientific (Monza, Italy).

Dimethyl fumarate (DMF) was obtained by Sigma-Aldrich (St. Louis, MO, United States). A selective p38 MAP kinase inhibitor SB202190 was purchased from Abcam (Cambridge, United Kingdom). All other chemicals and reagents were purchased by Sigma-Aldrich unless otherwise stated. The stock solution of DMF was prepared using DMSO and diluted with cell culture medium to obtain final dose for each treatment. As indicated, vehicle (0.2% v/v DMSO) corresponds to the amount present in the highest dosage and was used in all experiments to exclude unspecific and toxic effects.

All the reagents used were analytical grade or of the highest grade available. Dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium (DMEM) high glucose, methoxyamine hydrochloride (≥ 98%), N,O-bis(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane (BSTFA + 1% TMCS), sodium bicarbonate, sodium chloride (NaCl, ≥ 99.5%), trypan blue solution 0.4% (w/v), trypsin–EDTA solution, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl fumarate (DMF) and all standards used throughout the work were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). An antibiotic solution of 10,000 U mL⁻¹ penicillin/10,000 µg mL⁻¹ streptomycin, Hanks’ balanced salt solution (HBSS), and heat-inactivated fetal bovine serum (FBS) were obtained from Gibco Invitrogen (Barcelona, Spain). Methanol (≥ 99.9%), chloroform (≥ 99.8%), and pyridine (≥ 99%) were obtained from VWR (Leuven, Belgium). All sterile plastic material was obtained from Corning Costar (United States of America).

Dimethyl fumarate (DMF) and Hydrogen peroxide solution (H₂O₂, 30% w/w in H₂O, contains stabilizer) was purchased from Sigma–Aldrich (St. Louis, MO, USA). DMF was dissolved in DMSO and aliquots were kept in dark in −20 °C freezer. H₂O₂ was kept at 4 °C refrigerator. Newly opened bottle of H₂O₂ was used and freshly prepared and diluted H₂O₂ stock solution was immediately added into cell culture medium to achieve the desired working concentration. The concentration responses for both H₂O₂ (ranging from 0, 10, 20, 40, 80, and 100 μM) and DMF (ranging from 0, 10, 20, and 40 μM) and time course for both H₂O₂ and DMF (ranging from 4, 6, 18, 24, 48, and 72 h) were studied in the culture medium and then the optimal conditions (40 µm H₂O₂ and 10 µm DMF) for studying DMF neuroprotective effect under H₂O₂-induced oxidative stress were determined for subsequent experiments. In most experiments unless otherwise noted, the cultures were divided into four different groups which were treated in the following way: (1) Control with same concentration of DMSO used to dissolve DMF; (2) DMF alone; (3) H₂O₂ alone with same concentration of DMSO; (4) DMF + H₂O₂. At least three separate cultures were set up for each experiment.