Figure 3. Doxorubicin treatment enriches CSCs subpopulation and highlights that in CSCs p53 activity is attenuated and aberrantly localised within the cell.

(A) Cell viability of MACS-sorted J3T CD133+ and CD133- cells was assayed 48 hours after treatment with increasing doses of doxorubicin (* p < 0.005). (B) J3T cells were treated with the indicated doses of doxorubicin and the percentage CD133+ cells was determined 24 hours post-treatment. (C) CD133+ and CD133- cells were treated with doxorubicin and harvested 4 hours after treatment. Cell lysates were probed for the expression of phosphorylation of p53 at serine-15, MDM2, γH2AX and β-actin as a loading control. 30 μg was loaded per lane. (D) The subcellular localisation of p53, in J3T adherent cells and spheres, was determined 4 hours post-treatment with the indicated doses of doxorubicin by western blotting. Proteins were extracted according to their subcellular localisation: F1, cytosolic; F2, membranes/organelles; F3, nucleus; F4, nucleus; F5, cytoskeleton. 30 μg was loaded per lane. Coomassie staining (CM) confirmed that protein expression profiles from each fraction were distinct, 5 μg was loaded per lane.