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Figure 1 | Molecular Cancer

Figure 1

From: Downregulation of HuR as a new mechanism of doxorubicin resistance in breast cancer cells

Figure 1

A: HuR translocates from the nucleus to the cytosol after doxorubicin treatment. 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. An average of 300 cells for each experimental conditions were used. Z-score below zero indicates nuclear localization, above zero cytoplasmic localization. B: Subcellular fractionation on increasing doses of doxorubicin and quantification of HuR expression level. Western blotting on MCF-7 cells starved for 24 h and treated at increasing doxorubicin concentration (from 1 to 50 μM). The filter was probed with anti-H3 as nuclear fraction marker and anti-LDH as a cytosolic fraction marker. Quantification of total HuR protein measured by densitometric analyses on three independent western blots. C: HuR translocates from the nucleus to the cytosol after 1 and 4 h doxorubicin treatment. Immunofluorescence (HuR in green, counterstaining DAPI in blue) on MCF-7 cells starved or not (FCS) for 24 h and treated with doxorubicin 10 μM for 1 and 4 h. D: Subcellular fractionation after 1 and 4 h doxorubicin treatment. Western blotting on MCF-7 cells starved or not (FCS) for 24 h and with doxorubicin 10 μM for 1 and 4 h. The filter was probed with anti-H3 as nuclear fraction marker and anti-LDH as a cytosolic fraction marker. Quantification of HuR protein translocation after 10 μM doxo for 4 h measured by densitometric analyses on three independent western blots. * p-value < 0.01 with respect to starved conditions. E: Doxorubicin induces modification in HuR phosphorylation. 2D western blotting on whole cell lysates. MCF-7 cells were grown in standard condition (FCS), starved or in the presence of doxorubicin 10 μM for 4 h (doxo). As negative control for phosphorylation the doxorubicin sample was treated with calf intestinal alkaline phosphatase (CIAP). HuR immunoprecipitation blotted with anti HuR and pan anti-phospo antibody. As negative control IgG immunoprecipitation was blotted with anti HuR and pan anti-phospo antibodies. In: Input material, IgG: IgG immunoprecipitated material, HuR: anti-HuR immunoprecipitated material.

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