Sty bodipy

STY-BODIPY (1.5 μM) (cat No. 27089, Cayman Chemical Co, MI, United States) and liposomes of egg-PC (1 mM) (cat No. 840051P, Avanti Polar Lipids Inc, AL, United States) in TBS (pH 7.4) were added to an opaque 96-well polypropylene plate, followed by the addition of the indicated compounds (10 μM). The autoxidation was initiated by the addition of V-70 (0.5 mM) (cat No. 001-70078, Fujifilm Wako, Japan). For the enzymatic experiments, recombinant NQO1 (cat No. MBS201141, MyBioSource, CA, United States) (50 nM), recombinant FSP1 (cat No. 29611-50, Cayman Chemical) (50 nM), NADH (75 μM), the indicated quinones (5 μM), or a combination thereof, were added to the wells containing STY-BODIPY and liposomes prior to the addition of V-70. A lower compound concentration was used here to avoid rapid consumption of NADH by the most reactive quinones. Data were acquired by excitation of STY-BODIPY at 488 nm and emission was measured at 518 nm every 15 min at 37°C. Data were transformed into [ox-STY-BODIPY] by taking the raw fluorescence values of the saturated curve of control DMSO and dividing them by the initial concentration of reduced STY-BODIPY (1.5 μM). To obtain a unique number related to the data, the area under the curve (AUC) was calculated using Graph Pad Prism 6.

Bafilomycin (11038), BI-6C9 (17265), bisindoylmaleimide III (11072),
chlorgyline (15925), erastin (17754), trifluoromethoxycarbonylcyanide
phenylhydrazone (FCCP; 15218), (ferrostatin (17729), Flt 3 inhibitor (21193),
GKT137831 (17764), idebenone (15475), liproxstatin (17730), LY83583 (70230),
mitoquinol (mitoQ; 89950), Nullscript (16433), PD146176 (10010518), PI3K
inhibitor VIII (10009210), RSL3 (19288), Scriptaid (10572), STY-BODIPY (27089),
2-theonyl trifluoroacetone (TTFA; 15517) and troglitazone (71750) were purchased
from Cayman Chemical (Ann Arbor, MI USA). GSK2795039 (HX18950) was from MedChem
Express (Monmouth Junction, NJ USA). Cisplatin (2251) was from Tocris
(Minneapolis, MN USA). Antimycin A (A8674), (aminooxy)acetic acid
hemihydrochloride (AOA; C13408), apomorphine (A4393), CdCl₂ (202908),
8-(4-chlorophenylthio)-guanosine 3’,5’-cyclic monophosphate
(pCPT-cGMP; C5438), CoCl₂ (C2644), deferiprone (379409), glutamate
(G5889), glutaminase inhibitor 968 (SML1327), hydrogen peroxide
(H₂O₂; H1009), 6-hydroxydopamine (6OHDA; H4391),
iodoacetic acid (I4386), myxothiazol (T5580), paraquat (36541), rotenone
(45656), sodium azide (S8032), tert-butyl hydroperoxide (tBOOH; B2633) and all
other chemicals were purchased from Sigma-Aldrich (St. Louis, MO USA) unless
otherwise stated.