Histone Deacetylase Inhibitors Increase p27^Kip1 by Affecting Its Ubiquitin-Dependent Degradation through Skp2 Downregulation

Figure 3

Effect of BuA on the expression and turnover of . (a) K562 and EPN cells were treated with BuA 1 mM for different time intervals and then total RNA was prepared. Subsequently, the contents of (p27) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcripts were determined by RT-PCR (see Materials and Methods for experimental details). GAPDH was used as internal standard. (b) K562 cells were incubated for 24 hours with 1 mM BuA. Then, the nuclear and cytosolic compartments were prepared and content of CDK2 was determined by immunoblotting. Histone H3 and β-tubulin were used as markers of nuclear and cytosolic fraction, respectively, and for equal loading evaluation. (c) K562 cells were incubated without (Con) and with 1 mM BuA 1 for 16 hours. Then, the cells were pelleted and washed for removing BuA and 36 μM cycloheximide was added to the culture medium. Finally, cells were collected at different time intervals and analyzed for the content of (p27) by immunoblotting. (d) Three independent experiments as those reported in (c) were performed. The results of blots were analyzed and quantified by ImageJ64 software. The graph reports the average for each time period of these experiments and the bar the statistical variation.

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