Effect of AN-7 on the activity and expression of cellular HDACs classes I and II.

<p>(A) The cells were exposed to AN-7 for 3 h in the presence of the HDAC fluorogenic substrate. The average % of HDAC activity as a function of 0–100 µM concentration of AN-7 of a representative experiment conducted in quadruplicates is shown. (B) The average IC₅₀±SE of AN-7 for each of the cell types is calculated from three independent experiments. (C) Lysates of cells were loaded (35 µg protein/well) on 10% SDS gels and were subjected to Western blot analysis. HDAC1, HDAC2, HDAC5 or actin were detected with the appropriate antibodies. (D) HDAC activity of three experiments (in triplicate) conducted with 4T1 (cancer) and H9C2 (non-cancer) cells treated with AN-7 (50 µM) as a single agent for a total of 3 h, 1 h prior to the addition of the fluorogenic substrate and 2 h in its presence; Dox (200 nM) as a single agent treatment, or with the combination AN-7+Dox where 50 µM AN-7 was added 1 h prior to the addition of Dox and the fluorogenic substrate and then incubated for an additional 2 h. (E) The viability of the cells treated as described in (D) was determined by a Hoechst assay after 23 or 24 h for cells treated with Dox. (F) Cells were treated with AN-7 (3 or 24 h), Dox (2 or 23 h) or AN-7 (3 or 24 h)+Dox (2 or 23 h), as described (D). The lysates of the cells were subjected to Western blot analysis as described (C).