The 63 synthetic compounds that were used in the screen for inhibitors of the ESX–Sur2 interaction were provided by Professor Younghwa Na (College of Pharmacy, Cha University). These compounds have diverse core structures and include the following: 9 3-(3′-heteroatom substituted-2′-hydroxy-1′-propyloxy) xanthone analogues; 13 2,5,7-heteroatom substituted

chroman-4-one analogues; 13 benzosanthen-12-one derivatives; 12 4-hydroxy-2′-nitrodiphenyl ether analogues; 9 methyloxiranylmethoxyxanthone analogues; and 7 fluoroquinophenoxazine derivatives. Adriamycin, etoposide, buy PD0325901 camptothecin, canertinib and BMS599626 were purchased from Sigma–Aldrich (St. Louis, USA). Wrenchnolol was provided by Professor Uesugi (Kyoto University, Japan). All of the compounds used in the present study were dissolved in dimethylsulfoxide (DMSO; Sigma–Aldrich, St. Louis, USA) to form 10 mM stock solutions and stored at −20 °C until needed. Human breast cancer cell lines (MCF-7, MDA-MB231, T47D, SK-BR-3) and a human kidney cell line (HEK293) were purchased from the Korean Cell Line Bank (Seoul, Korea). AU-565 (human breast adenocarcinoma cell line) and MDA-MB468 (human breast cancer cell line) were kind gifts from Dr. Seung Bae Rho (National Cancer Center, Korea) and Dr. Yung-Jue Bang (College of Medicine,

Seoul National University, Korea). All cell lines except HEK293 were maintained in Roswell Park Memorial Institute Medium (RPMI 1640, WelGENE Inc., Daegu, Korea) that was supplemented with 10% fetal bovine serum (FBS, WelGENE Inc. Daegu, Korea) and 1% penicillin–streptomycin (Hyclone laboratories GDC-0199 ic50 Inc., Rockford, IL, USA). HEK293 was cultured in Dulbecco’s Modified Eagle Medium (DMEM, WelGENE Inc., Korea) with 10% FBS and 1% penicillin–streptomycin. These cells were grown

at 37 °C in a humidified atmosphere containing 5% CO2. The cells were seeded in 96-well microplates at a density of 1–2 × 104 cells per well and incubated overnight in 0.1 mL of medium supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C in a Resminostat 5% CO2 incubator. On day 2, after 4 h of FBS depletion, the compounds were treated by exchanging the media with 0.1 mL aliquots of medium containing graded concentrations (0, 0.1, 0.25, 0.5, 1, 2 and 5 μM as a final concentration). After 48 h of treatment, 5 μL of cell counting kit-8 (Dojindo, Kumamoto, Japan) was added to each well followed by an additional 4 h of incubation under the same conditions. The absorbance of each well was determined using an Automatic Elisa Reader System (Bio-Rad 3550, Ramsey, MN, USA) at a wavelength of 450 nm. The viability of cells treated with CHO10 was calculated from the absorbance, with untreated cells assumed to be 100% viable. The cells were seeded in 60 mm dishes at a density of 5 × 105 cells per dish and incubated until the cells reached a confluence of 80%.