Figure 4.

Translocation and decreased expression of Skp2 in senescent prostate cancer cells after treatment with multiple senescence-inducing drugs. Band intensities are normalized to α-tubulin and expressed in relative units. (A) Immunoblot analysis of p27^Kip1 and its relationship to Skp2 in proliferating DU145 and PC3 cells induced to senescence by 250 nM AZQ. Cells were exposed to DMSO (-) or AZQ (+) for 3 days with 3 days of recovery in drug-free medium before harvesting. (B) Time course experiments in PC3 using multiple senescence-inducing agents. Immunoblot analysis of p27^Kip1 and Skp2 expression in untreated PC3 cells (Control) harvested at multiple time points after adding the senescence-inducing drugs AZQ, doxorubicin (DOX), or 5-AZA. Treated PC3 cells were harvested after 2 and 3 days of drug exposure and after 3 days of incubation with drug followed by 3 days in drug-free medium (Removal + 3 Days). p27^Kip1 expression is induced before Skp2 down-regulation with all drugs analyzed. (C) Immunofluorescence microscopy of p27^Kip1 (red) and Skp2 (green) over time during TIS in PC3 cells. Cells were fixed and stained after incubation with AZQ as above. Nuclei were stained with Hoechst 33342 (blue). Scale bar, 20 µm.