The PCR products underwent electrophoresis on a 1.2% agarose gel to analyze the expression level of the HER2 gene. The primers used for HER2 were as follows: forward 5′-GAGCACCCAAGTGTGCAC and reverse 5′-TTGGTTGTGAGCGATGAG. AZD9291 order SK-BR-3 cells were seeded in 60 mm dishes at a density of 5 × 105 cells per dish. When the cells reached a confluence of 80%, the cells were treated with the compounds at the concentrations indicated in the figure legends. Subsequently, the cells were washed with ice-cold PBS (pH 7.4) and harvested by centrifugation at 2000 rpm for 5 min. The cell pellet was fixed with 70% ethanol. The fixed cells were washed with PBS before incubation with 50 μg/mL of propidium iodide (Sigma, St. Louis, MO, USA) and

2.5 μg/mL of RNase (Sigma, St. Louis, MO, USA). Fluorescence was measured with a Fluorescence-Activated Cell Sorting (FACS)-Caliber flow cytometer (BD Biosciences, Lakes, NJ, USA). At least 10,000 cells were measured for each sample. HEK293T human

kidney cells BYL719 manufacturer were seeded in 96 well microplates at a density of 5 × 103 cells per well and incubated overnight. Mammalian expression vectors encoding the activation domain of ESX, which were fused to the GAL4 DNA-binding domain (amino acids 1–94), were co-transfected into HEK293T cells at a range of concentrations for each individual compound with a reporter plasmid, as previously described (Shimogawa et al., 2004). The reporter plasmid of the IL2 promoter carried five GAL4 binding sites that produced secreted alkaline phosphatase (SEAP) in an amount proportional to the interaction between GAL4-ESX and endogenous Sur2, which PAK6 is a subunit of the human mediator complex. After 12 h of treatment with each compound, a 40 μL aliquot of culture medium was incubated at 65 °C for 3 h to inactivate all of the endogenous enzymes except for the SEAP enzyme. The 4-methylumbelliferyl phosphate (MUP) solution, which is a fluorescent SEAP substrate, was added to each well and incubated at 37 °C for at least 3 h in the dark. After incubation,

the SEAP activity was measured with a Microplate Fluorescence Reader (SpectraMAX GEMINI EM, Molecular Devices, Sunnyvale, CA, USA) using an excitation wavelength of 360 nm and an emission wavelength of 440 nm. To verify that the signal decrease was caused by the compounds’ inhibitory activity against the ESX–Sur2 interaction and not by cell death, 5 μL of WST-1 (Promega, Madison, WI, USA) was added to each well of the remaining cell culture after removal of the aliquot for the SEAP assay. This solution was incubated at 37 °C for at least 2 h. After incubation, the absorbance of each well was measured with an Automatic Elisa Reader System (Bio-Rad 3550, Hercules, CA, USA) at a wavelength of 450 nm. Kinase inhibitory activities of CHO10 were evaluated using the Millipore kinase profiling services with HER1, HER4, IGF1R, MAPK1 and MAPK2 kinases, following the KinaseProfiler Service Assay protocols.