A. Characterization of MCF-7/DoxoR cells. Western blotting on whole cell lysates from parental (MCF-7) and doxorubicin resistant (MCF-7/DoxoR) MCF-7 cells. HuR, c-Myc, Socs3 and TOP2A are down regulated in the MCF-7/DoxoR cells in comparison with the parental, ABCG2 is up regulated. Caspase 7 expression level is not affected. Beta tubulin is the loading control. B. Characterization of MDA-MB-231/DoxoR and SK/NOdoxoR cells. Western blotting on whole cell lysates from parental (MDA-MB-231, SK) and doxo resistant (MDA/DoxoR) and doxo exposed (SK/NOdoxoR) cells. HuR and TOP2A are down regulated in the MDA-MB-231/DoxoR cells. Beta tubulin is the loading control. Protein quantification measured by densitometric analyses on three independent western blots. * p-value < 0.05 with respect to starved conditions. C. HuR does not translocate from the nucleus to the cytosol after doxorubicin treatment in MCF-7/DoxoR cells. Immunofluorescence (HuR in green, counterstaining DAPI in blue) on MCF-7 cells starved for 24 h and treated at increasing doxo concentration (from 1 to 50 μM) for 4 h. Quantification of HuR cytoplasmic translocation measured on immunofluorescence images by calculating the ratio of intensity signal in the nucleus and in the cytoplasm. D. An average of 300 cells for each experimental conditions were used. Z-score below zero indicates nuclear localization, above zero cytoplasmic localization. Quantification of HuR protein translocation after 10 μM doxo for 4 h measured by densitometric analyses on three independent western blots. E. Quantification of total HuR protein measured by densitometric analyses on three independent western blots. No change was observed neither in protein amount nor in protein translocation during doxo treatment.
Latorre et al. Molecular Cancer 2012 11:13 doi:10.1186/1476-4598-11-13