The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Invitrogen, Carsbad, CA, USA) containing 10% heat inactivated fetal bovine serum (HyClone, Logan, UT, USA) at 37°C in humidified 95% air/5% CO2 incubator. When the cultures reached confluence, subculture was prepared using a 0.02% EDTA-0.05% trypsin solution. The cells were grown on well tissue culture plates and used 1-2 days after plating when a confluent monolayer culture was achieved.

Unless otherwise stated, cells were treated with Savolitinib in vivo silibinin in serum-free medium. Test reagents were added to the medium 30 min before silibinin exposure. Selleck Cediranib Measurement of cell viability Cell viability was evaluated using a MTT assay [9]. Culture medium containing 0.5 mg/ml of MTT was added to each well. The cells were incubated for 2 h at 37°C, the supernatant was removed and the formed formazan crystals in viable cells were solubilized with 0.11 ml of dimethyl sulfoxide. A 0.1 ml aliquot of each sample was then translated to 96-well plates and the absorbance of each well

was measured at 550 nm with ELISA Reader (FLUOstar OPTIMA, BMG LABTECH, Offenburg, Germany). Data were expressed as a percentage of control measured in the absence of silibinin. Measurement buy Ganetespib of calpain activity Calpain activity was measured by calpain assay kit (BioVision Research Products, CA, USA) according to the manufacturer’s instructions. Cells were grown in 6-well plates and were treated as indicated. Detached cells from the bottom of culture plates by trypsin were pelleted by centrifugation and washed with phosphate-buffered saline (PBS). The pellet were suspended in extraction buffer and incubated on ice for 20 min then centrifuged at 10,000 × g for 10 min at 4°C. The supernatant represented the cytosolic protein. Add 10 μl of 10× reaction buffer and 5 μl of calpain substrate, Ac-LLY-AFC, to each assay. Incubate at 37°C for 1 h in the dark. After incubation, production of free AFC was fluorometrically measured suing a Victor 3 Multilabel Counter with

an excitation filter of 400 nm and an emission filter of 505 nm (PerkinElmer, Carbohydrate Boston, MA, USA). Measurement of reactive oxygen species (ROS) The intracellular generation of ROS was measured using DCFH-DA. The nonfluorescent ester penetrates into the cells and is hydrolyzed to DCFH by the cellular esterases. The probe (DCFH) is rapidly oxidized to the highly fluorescent compound DCF in the presence of cellular peroxidase and ROS such as hydrogen peroxide or fatty acid peroxides. Cells cultured in 24-well plate were preincubated in the culture medium with 30 μM DCFH-DA for 1 h at 37°C. After the preincubation, the cells were exposed to 30 μM silibinin for various times. Changes in DCF fluorescence was assayed using FACSort Becton Dickinson Flow Cytometer (Becton-Dickinson Bioscience, San Jose, CA, USA) and data were analyzed with CELLQuest Software.