Effect of HDACi on cell cycle, p21 expression, bak expression and Sp1 expression and acetylation. The extent of the concomitant response of Sp1 acetylation, cell cycle arrest and p21 up-regulation was determined using a high-content biology approach. HCT116 cells were treated with concentration ranges of 0-20 mM sodium butyrate, 0-20 mM valproic acid (VPA), 0-20 μM Oxamflatin, 0-20 μM Scriptaid, 0-20 μM APHA compound 8, 0-20 μM CHAHA. In all cases treatments were carried outfor 24 h. Cells were stained using immunocytochemistry for DNA content (Hoescht), p21, bak, Sp1 and acetyl-Sp1 as described in the methods section. Cells were analysed, on the basis of DNA content, for cell cycle phase and divided into G1 (filled circles), S (filled squares) or G2/M (filled triangles). Levels of protein were calculated from mean total fluorescence and are expressed in terms of fluorophore fluorescence relative to that observed in untreated cells. Sp1 (filled squares) and acetyl-Sp1 (open squares) are shown on the same graph.