Kinase inhibitors

Kinase inhibitors were purchased from Selleck Chemicals and were diluted in DMSO then added to the cells at the concentrations indicated within each figure. For determining IC50 values across the range of Kinase inhibitors, compounds were serially diluted 1:2 to produce eleven concentrations, with the highest concentration being 10 μM. The final concentration of DMSO in the wells was 0.5%. Positive and negative control wells were included for each plate where the cells were treated with 1 uM of Erlotinib (Selleck Chemicals) or 0.5% DMSO, respectively. For the hypoxia-treated cells, cells grown under normoxic conditions were used as negative controls. The cells were grown in a humidified atmosphere at 37°C/5% CO₂ for 72 h. Hypoxia-treated cells were grown in a hypoxic chamber at 37°C/5% CO₂ with 1% O₂ for 72 h.

Titanium dioxide (TiO₂, titania) particles (Titansphere, particle size: 10 μm), Empore SDB-XC and C8 membrane disks were obtained from GL Sciences (Tokyo, Japan). Sequencing-grade modified trypsin was purchased from Promega (Madison, WI, USA). Recombinant protein kinases were obtained from Carna Biosciences (Kobe, Japan). Kinase inhibitors were purchased from Selleck Chemicals (Houston, TX, USA). Water was purified by a Millipore Milli-Q system (Bedford, MA, USA). Alkaline phosphatase from calf intestine, DL-lactic acid, MS-grade Lys-C (lysyl endopeptidase), and all other chemicals were purchased from Fujifilm Wako (Osaka, Japan).

MCF7, MDA-MB-231 and COS7 cell lines were obtained from the ATCC and maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS, Eurobio). All cell cultures were carried out at 37°C using a 5% CO2 atmosphere. For cell stimulation studies, cell lines were stimulated with Sodium pervanadate (PV, premix of 1 mM H₂O₂ and 1 mM Na₃VO₄) and incubated for 15 min at 37°C. For the evaluation of the effect of the kinase inhibitors (all from Selleckchem), cells were incubated in medium for 2 hours with 2.5 mM R406, 5 mM PRT062607, 1 mM PP2, 0.5 mM AZD0530. Stock solutions for all those kinase inhibitors were prepared in dimethyl sulfoxide (Sigma, Hybri-Max grade), which is used as vehicle negative control

The TNBC cell lines, MDA-MB-231 and MDA-MB-468, were purchased from ATCC (Manassas, VA, USA). They were authenticated by ATCC by short tandem repeat profiling, cell morphology monitoring, karyotyping, and cytochrome C oxidase I testing. The cells were grown in ATCC-recommended media, containing 10% FBS and 1% Penicillin-streptomycin (Thermo Fisher Scientific, Pittsburgh, PA, USA). All cells were cultured at 37 ℃ in humid atmosphere containing 5% CO₂. Kinase inhibitors were purchased from Selleckchem (Munich, Germany), LC Laboratories (Woburn, MA, USA), or AdooQ Bioscience (Irvine, CA, USA).

All CRC cell lines were purchased from ATCC (Manassas, VA, USA), and were used within 6 months of their purchase. ATCC authenticates all its cell lines by short tandem repeat profiling, cell morphology monitoring, karyotyping, and cytochrome C oxidase I testing. The cells were grown in ATCC-formulated McCoy’s 5a Medium Modified (Catalog No. 30-2007), RPMI-1640 Medium (Catalog No. 30-2001), or Eagle’s Minimum Essential Medium (Catalog No. 30-2003), containing 10% FBS and 1% penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA, USA). All cells were cultured at 37 °C in humid atmosphere containing 5% CO₂. Kinase inhibitors were purchased from Selleckchem (Munich, Germany), LC Laboratories (Woburn, MA, USA) or AdooQ Bioscience (Irvine, CA, USA).