Wst 8 colorimetric reagent

HUVECs (5 × 10⁵ cells/vial) was purchased from Kurabo Inc. and grown in HuMedia-EG2 medium with growth supplements (Kurabo Inc., Osaka, Japan). HUVECs in the culture medium was plated into each well of 96-well plates (2 × 10³ cells/well/100 µL). After 24 h, the serially diluted compounds, which were dissolved in the medium containing no more than 0.5% EtOH, were added, and then the plates were incubated for an additional 72 h in a humidified atmosphere of 5% CO₂ at 37 °C. The cell proliferation was detected by WST-8 colorimetric reagent (Nacalai Tesque, Inc., Kyoto, Japan). The IC₅₀ value was determined by linear interpolation from the growth inhibition curve.

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).

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. The Mito Check Complex Activity Assay Kit, used to evaluate the effect of test samples on mitochondrial complexes I–V, was obtained from Cayman Chemical (Ann Arbor, MI, USA). The oxygen utilization of cells and mitochondrial membrane potential were measured by using an Oxygen Consumption Rate Assay Kit from Cayman Chemical and JC-1 MitoMP Detection Kit from Dojindo Laboratories (Kumamoto, Japan), respectively. Rotenone, thenoyltrifluoroacetone (TTFA), carbonyl cyanide m-chlorophenylhydrazone (CCCP), antimycin A, and oligomycin mixture were purchased 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, respectively.