Mitochondrial Hydrogen Sulfide Donor Synthesis

AP39, a novel mitochondria-targeted H₂S donor, was designed and synthesized by Medicilon Inc., as described previously [19 (link), 20 (link)]. Antimycin A, 0.25% trypsin, 2-deoxyglucose, oligomycin, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), trichloroacetic acid, rotenone, 7-azido-4-methylcoumarin (AzMC), monobasic potassium phosphate, dibasic sodium succinate hexahydrate, adenosine 5′-triphosphate (ATP) disodium salt hydrate, fatty acid-free BSA, sodium hydrosulfide hydrate (NaSH·xH₂O), N,N-diethyl-p-phenylenediamine sulfate, magnesium chloride, and iron(III) chloride (FeCl₃) were purchased from Sigma-Aldrich Company. The monoclonal anti-Aβ antibody 6E10 was obtained from Covance Inc. (Princeton, NJ, USA). Drp1 was purchased from Novus Biologicals, Inc. Fis1 was purchased from Proteintech Group. Mfn1 and Mfn2 antibodies were purchased from Santa Cruz Biotech, and OPA-1 antibody was purchased from BD Transduction Laboratories. The secondary antibodies goat anti-mouse-HRP and donkey anti-rabbit-HRP antibodies were purchased from GE Healthcare. The Pierce BCA protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco's modified Eagle's medium (DMEM), 1% glutamine, and 1% penicillin were obtained from Invitrogen. All other chemicals were purchased from Amresco (Solon, OH, USA).