Fig. 1

Generation of ccRCC cell lines adapted to grow in the presence of sunitinib. (A) Phase-contrast microscopy pictures of live cells, i.e. 786-O, 786-O-SR (adapted to grow in the presence of 8 µM sunitinib), A-498 and A-498-SR (adapted to grow in the presence of 7 µM sunitinib). Sunitinib-resistant (SR) cells show hypertrophic features indicated by their increased size as compared to the corresponding parental cell lines. Pictures were taken at 10x magnification. Scale bar = 100 μm. (B) The effect on cell growth of increasing concentrations of sunitinib (Sun) was analysed by the IncuCyte S3 live-cell system. Measurements were performed every four hours. Data represent mean values +/- STDEV (n = 9 replicates) and expressed in percentage. Control cells were grown in the presence of DMSO. Control^* denotes cells adapted to grow in the presence of 8 µM sunitinib (786-O-SR) or 7 µM sunitinib (A-498-SR). (C) Analysis by Western blot of whole cell lysates from 786-O, 786OSR, A-498 and A-498-SR cell lines. Harvested cells were processed as described in the materials and methods section and whole cell lysates were analysed by probing Western blot membranes with antibodies directed against the indicated proteins. Western blot band intensity of the indicated proteins was determined using ImageJ densitometry analysis (NIH). Values are expressed in percentage. All experiments were performed at least three times and yielded similar results. One representative experiment is shown. β-actin detection served as loading control