Effect of imatinib on sis-NIH3T3 cell viability. Growth factors included in serum such as insulin-like growth factor-I (IGF-I) (Gibco), basic dapt secretase growth factor (bFGF) (PeproTech, Rocky Hill, NJ, USA), PDGF-AB (PeproTech), epidermal growth factor (EGF) (Upstate Biotechnology, Lake Placid, NY, USA) and transforming growth factor beta1 (TGFβ1) (PeproTech) were tested with regard to rescue capability of imatinib-induced cell death in sis-NIH3T3 fibroblasts. Confluent cultures of sis-NIH3T3 fibroblasts were exposed to growth factors with or without imatinib (Glivec, kindly provided by Novartis) in serum-free conditions for 24 h. Then, the cell viability was monitored using WST-8 (Dojindo, Kumamoto, Japan) colorimetric assay. After the WST-8 assay, the cells were stained with crystal violet, and photographed under an inverted microscope.
Apoptosis assay. To examine whether imatinib-induced sis-NIH3T3 cell death is caused by apoptosis, Bax/Bcl-2 ratio was determined by quantitative real-time RT-PCR [22] at several time points, indicated in the figure legend, using SybrGreen [23]. Bax and Bcl-2 primer sequences are shown in Supplementary Table 1. A DNA fragmentation assay using a DNA ladder isolation kit (Calbiochem, Merck, Darmstadt, Germany) was also carried out after 24 h.
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Such low level of genetic differentiation
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