Interestingly, resistance to imatinib in the KR cells is through a BCR/ABL-independent manner

mM sodium pyruvate, and 4 mMGlutaMAX was used. Inhibition of OXPHOS was achieved by adding 2.5 mM oligomycin to normal culture medium (containing 25 mM glucose). For live imaging, phenol red-free media were prepared using DMEM Base powder from SigmaAldrich to which sodium bicarbonate, GlutaMAX, sodium pyruvate, glucose and/or galactose, 2-DG, or oligomycin were added. Materials and Methods Reagents All reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA), unless stated otherwise. PLOS ONE | www.plosone.org 2 Proliferation Assay For cell proliferation analysis, the protocol developed by Skehan et al. [49] was used. RAW 264.7 cells were seeded in four 96-well plates (30,000 cells/well) in 100 ml culture medium and incubated May 2014 | Volume GW 501516 chemical information PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19653943 9 | Issue 5 | e96786 Glucose Controls Macrophage Morphodynamics for eight hours. At T0, plates were washed once and the medium in plates T6, T15, and T24 was replaced with either control, galactose, gluc/gal, 2-DG, or oligomycin medium containing 100 ng/ml LPS, while plate T0 was fixed for sulforhodamine B (SRB) staining of protein content. After 0, 6, 15, and 24 hours, cells were washed twice with cold PBS and fixed with 10% trichloroacetic acid (TCA; J.T.Baker, Deventer, Holland) for 1 hour at 4uC. After fixation, plates were washed five times with water and stored at 220uC until all plates were collected. Cellular protein was stained with 50 ml 0.5% SRB in 1% acetic acid for 20 minutes after which wells were washed four times with 1% acetic acid. Plates were dried at 60uC for 3 hours, protein was dissolved in 150 ml 10 mM Tris-HCl (pH 10.5), and the absorbance of each well was measured at 510 nm on a BioRad Benchmark Plus micro plate reader. Values were corrected for backg