Ku60019

Stock solutions of NVP-BEZ235, RAD001, NVP-BKM120 and GSK2334470 (Selleckchem, Munich, Germany), MK-2206 and INK128 (Active Biochem, Bonn, Germany), PIK-90 (Merck Chemicals GmbH, Darmstadt, Germany), NU7441 and KU60019 (Tocris Bioscience, Bristol, United Kingdom) were prepared in DMSO. Working concentrations were freshly prepared in medium with control corresponding to highest DMSO concentration.

Cells, in logarithmic growth, were treated with a titration series of 10 to 0.01 μM ATM inhibitor (ATMi) (KU 60019, Tocris, Bristol, UK), CHK1 inhibitor (CHK1i) (PF-477736, Merck, Molsheim, France), CHK1/2 inhibitor (CHK1/2i) (AZD-7762, Merck, Molsheim, France), Wee1 (Wee1i) (Adavosertib, MedChemTronica, Stockholm, Sweden), or p21 (p21i) (UC2288, Merck, Molsheim, France) diluted in dimethyl sulfoxide (DMSO). After 72 h a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed where 20 μL CellTiter 96^® AQueous One Solution Assay (Promega, Stockholm, Sweden) was added per 100 μL media. The plates were then incubated for 3 h and analysed in the microplate reader FLUOstar Omega (BMG LABTECH, Ortenberg, Germany). The absorbance at 490 nm and 690 nm was measured to assess the cell viability post-treatment. Each treatment was completed in triplicates. DMSO was used as a negative control.

The following inhibitors were used: Z-vad FMK (Promega, 100 μM), Q-VD-OPh (Sigma, 20 μM), PV-1019 (Merck-Millipore,10 μM), CHK2 inhibitor II (Merck-Millipore,10 μM), NU-7441 (Adooq, 20 μM), KU60019 (Tocris, 10 μM). All inhibitors were added to cells prior to nucleofection or irradiation and maintained in the growth medium for the experiment duration.

CIN612 cells were treated with or without ATM-kinase inhibitor KU-60019 (10uM, Tocris, Bristol, UK, Cat. #4176) or Caffeine (100mM Stock) (Sigma, Cat. # C0750) or their respective vehicle (DMSO, PBS) in E-medium supplemented with mouse epidermal growth factor (EGF) for the indicated times as previously described [26 (link)],[51 (link)]. The cells were subsequently harvested for protein.

Prior to treatment, primary HFMs were incubated for 24 h in ‘starved’ 2:1 melanocyte media (2:1 melanocyte media minus 1% FBS and BPE). All treatments were carried out in starved 2:1 melanocyte media for the duration of experiments. Pro-oxidants: fresh hydrogen peroxide (Sigma, UK) solution was prepared as a 10 mM stock solution immediately before use then diluted to the required concentration. Menadione (Sigma, UK) was dissolved in DMSO to 100 mM then diluted to 100 µM in media immediately prior to use. Antioxidants: Vitamin E and Quercetin (VitE/Q; Sigma, UK) were dissolved in water (1 mM) and DMSO (10 mM) respectively before combining to a final concentration of 10 µM (Vitamin E) and 1 µM (Quercetin) in media. Cells were pre-incubated with VitE/Q for 1 h prior to ROS induction. The ATM specific kinase inhibitor KU60019 (Tocris, UK) was dissolved in DMSO to 100 mM then diluted to 5 µM directly into media. Cells were pre-incubated in KU60019 for 1 h prior to ROS induction.

Mirin and KU-60019 were both purchased from Tocris, Bristol, UK. Mirin is a small-molecule inhibitor that blocks the 3ʹ and 5ʹ exonuclease activity associated with Mre11 and prevents ATM activation in response to double-strand breaks (Dupre et al., 2008 (link); Stivers, 2008 (link); Garner et al., 2009 (link)). KU-60019 is a potent ATM kinase inhibitor and has been used to inhibit migration and invasion of human glioma cells in vitro (Golding et al., 2009 (link)).