Rotenone

L-α-phosphatidylcholine (95%) and fluorescent lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (DOPE-RhB, >99%) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). Cholesterol (≥99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Homologous series of IA-n(OH) (n = 10, 12, 14, 16) and TPPB-n (n = 10, 12, 14) amphiphiles were synthesized according to the published methods [70 (link),71 (link)]. Hexadecyltriphenylphosphonium bromide (TPPB-16, ≥98%) was purchased from Alfa Aesar (Haverhill, MA, USA). Rotenone (>95%) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). MitoTracker Green FM (98%) was used to stain mitochondria of living cells (Thermo Fisher Scientific, Waltham, MA, USA). For cellular uptake assay, 4′,6-diamidino-2-phenylindole (DAPI) was used (Sigma-Aldrich, St. Louis, MO, USA). Sodium phosphate buffer (PBS) was purchased from UralChemInvest (Ufa, Russia). Chloroform and ethanol (HPLC) were purchased from JSC №1 BASE Chemical reagents (Staraya Kupavna, Russia). Liposomal dispersions were prepared using ultrapure Milli-Q water purified by Simplicity^® UV system (Millipore SAS, Molsheim, France).

Dulbecco’s Modified Eagle’s medium (DMEM), WST-8 colorimetric reagent, and KCN were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Fetal bovine serum (FBS) and Dialyzed FBS were purchased from Equitech-Bio Inc. (Kerrville, TX, USA) and Thermo Fisher Scientific Inc. (Waltham, MA, USA), respectively. Anti-Akt, Anti-phosphorylated Akt, anti-GRP78, and anti-β-actin antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Horseradish peroxidase (HRP)-linked anti-rabbit IgG antibody (GE Healthcare Life Sciences, Buckinghamshire, UK) was used as secondary antibody. Mito Check Complex Activity Assay Kit (Cayman Chemical, Ann Arbor, MI, USA) was used to evaluate the effect of compound 1 on the mitochondrial complex I–V. Oxygen consumption of cells was measured by Oxygen Consumption Rate (OCR) Assay Kit (Cayman Chemical, Ann Arbor, MI, USA). Rotenone, thenoyltrifluoroacetone (TTFA), carbonyl cyanide m-chlorophenylhydrazone (CCCP), antimycin A, and oligomycin A were obtained from Tokyo Chemical Industry Co., LTD. (Tokyo, Japan), Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Sigma-Aldrich (St. Louis, MO, USA), LKT Laboratories, Inc. (St. Paul, MN, USA), and Cayman Chemical (Ann Arbor, MI, USA), respectively. Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Kishida Chemical Co., Ltd. (Osaka, Japan).

Mouse primary Müller cells, prepared as described previously, and mouse brain-derived microglia (BV-2) were maintained in DMEM10. Müller and BV-2 cells were seeded at 3.0 × 10⁵ and 6.5 × 10⁵ cells in 60 mm dishes, respectively, and cultured in media containing various concentrations of rotenone (Tokyo chemical industry, Tokyo, Japan) for 24 h. Cells were washed with DMEM10 twice to remove rotenone and were then cultured in fresh media for an additional 24 h. Culture media were collected and passed through a 0.22 µm filter (Merck Millipore, Burlington, MA, USA). To obtain double-conditioned media, Müller and BV-2 cells (1.4 × 10⁵ and 3.0 × 10⁵ cells, respectively) were cultured with rotenone-stimulated BV-2 and Müller cell-conditioned media (3 mL) in 35 mm dishes for 24 h, respectively.