Figure 2

A: HuR is necessary for caspase 3/7 activation doxorubicin induced. Histogram shows the caspase 3/7 activation (as luciferase intensity) in MCF-7 cells in the presence of increasing doxorubicin concentration with (light grey bar) or without (dark gray bar) HuR silencing. Graph of a representative experiment out of three experiments. Experiments were run in quadruplicate. * p-value < 0.01 with respect to HuR silenced cells. B: HuR silencing. Immunofluorescence on MCF-7 cells at 24 h after the siRNA HuR transfection. HuR is represented in green, counterstaining DAPI is blue. C: Doxorubicin induced HuR phosphorylation is blocked by rottlerin. 2D gel western blots on whole MCF-7 cell lysates cells treated with Rottlerin 10 mM for four hours and in co-administration with doxo. HuR immunoprecipitation blotted with anti HuR and pan anti-phospo antibodies. 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. The drug was tested for its capacity to modify HuR phosphorylation status alone or together with doxorubicin (doxo). Control for doxo alone is shown in Figure 1E D: HuR localization. representative immunofluorescence images of HuR localization (green) in control conditions (FCS, starvation, doxo) and in the presence of Rottlerin and with pre-treatment with one hour Rottlerin and four hours doxo. Images indicate that Rottlerin blocks doxo induced HuR translocation. E. Apoptotic response activation of MCF-7. Histogram shows the caspase 3/7 activation (as luminescent intensity) of MCF-7 cells in the presence of rottlerin (light grey) or rottlerin with doxorubicin (dark gray). Graph of a representative experiment out of three experiments. Experiments were run in quadruplicate. NS, Not Significant with respect to control (CTRL), * p-value < 0.01 with respect to doxorubicin treatment. F. Rottlerin blocks PMA induced PKCδ activation. representative confocal immunofluorescence images of PKCδ localization (green) in control conditions (FCS, rottlerin, PMA) and in the presence of coadministration of Rottlerin and PMA. Images indicate that Rottlerin blocks PMA induced PKCδ membrane translocation, known mechanism of PKCδ activation.