Mitoquinone mesylate

CAI was synthesized by the Institute of Materia Medica, Chinese Academy of Medical Sciences (Beijing, China). l-Glutamine-¹⁴ (link)C_5, rotenone (RTN) and 3-nitropropionic acid (3-NPA) were purchased from Sigma–Aldrich (MO, USA). V-9302 and CB-839 were purchased from Selleck Chemicals (CA, USA). Mitoquinone mesylate, Mito-TEMPO and R162 was purchased from MedChemExpress (NJ, USA). MitoTracker Red, BODIPY FL C16 (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid), 2-NBDG and MitoSOX Red were purchased from Thermo Fisher (MA, USA).

Human CD4⁺ and CD8⁺ T cells were isolated from the PBMCs of patients with RRMS and healthy donors by immunomagnetic separation using CD4⁺ or CD8⁺-labelled magnetic-activated cell sorting (MACS) microbeads (Miltenyi Biotec), respectively. For the purification of the T-cell subsets [memory (T_M) and naïve (T_N)], CD4⁺ or CD8⁺ T cells were enriched using a negative selection technique (Miltenyi Biotec) and subsequently T_M cells (CD45RO⁺) and T_N cells (CD45RO⁻) were isolated using CD45RO⁺-labelled MACS microbeads. All T cells were cultured in X-VIVO^TM Media 15 (Lonza). When indicated, 5 µg/ml DMF, 5 µg/ml monomethyl fumarate, 250 nM N-acetylcysteine (NAC), 250 µM glutathione (all from Merck) and 100 µM mitoquinone mesylate (MitoQ; MedChem Express) were used. Sulphoraphane (500 nM, Merck) was applied for pharmacological activation of Nrf2.²³ (link) For pharmacological inhibition of Nrf2, 5 µg/ml ML385 (Merck) was used.²⁴ (link) Buthionine sulphoximine (3 mM, Merck) was utilized as a specific inhibitor of glutamate cysteine ligase, the rate-limiting step in glutathione synthesis.²⁵ (link) For a detailed description see the Supplementary material.