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

Figure 5

Effect of HDACIs on Skp2 content. (a) EPN and K562 cells were treated with 1 mM BuA for the indicated time periods. Then, cellular extracts were prepared and Skp2 was determined by immunoblotting. Actin was evaluated for confirming equal loading. Con, untreated cells. (b) On the left, K562 cells were incubated for 24 hours with different HDACI at the concentrations indicated in Figure 1(a). Then, cellular extracts were prepared and analyzed for Skp2 and actin content by immunoblotting. On the right, K562 cells were cultured in the presence of BuA and NAM (as in Figure 1(a)). Then, cellular extracts were prepared and analyzed for Skp2 and actin content by immunoblotting. Con, untreated cells. (c) Various cell lines (RM, UT-7, Lan-5, HeLa, K562, and JURL-MK1) were cultured with 1 mM BuA for 24 hours. Then, Skp2 and actin were determined by immunoblotting. Con, untreated cells. (d) K562 cells were incubated with BuA for different time periods (as reported). Then, total RNA was prepared, retrotranscribed, and employed for evaluating Skp2 mRNA by quantitative PCR. The experiment was performed in triplicate. Con, untreated cells. and compared to Con. ns, not significative with respect to Con. (e) K562 cells were treated with 1 mM BuA for 16, 24, and 48 hours. Then, extracts were prepared and Skp2 was determined by immunoblotting. Actin was evaluated for confirming equal loading. Con, untreated cells.

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