Fig. 3

Effect of HDACis ± HY-PDT on HDAC expression and histone acetylation. a mRNA (by qRT-PCR) and b, c protein levels (by Western blotting) were measured in HT-29 cells after a sequential treatment starting with HDACis (for 24 h) followed by activation with hypericin for 8 h (a–c). Samples treated with drug-free vehicle solvents (<0.1% DMSO) were used as the reference control (Ct). Values are expressed as fold of Ct and represent the average ± SD of three independent experiments each done in triplicates. HDAC mRNA levels were normalized relative to those of the housekeeping gene, GAPDH. b HDAC and c histone H3 acetylation protein levels were quantified by densitometry, normalized relative to the loading controls (beta-actin and histone H3, respectively), and expressed as folds of the reference Ct. Western blots are representative of two independent experiments. Data was analyzed using one-way ANOVA with Tukey post hoc test. All conditions were compared to the reference Ct (*p < 0.05, **p < 0.01, ***p < 0.001), and the combined treatments were compared to HY-PDT alone (ǂp < 0.05, ǂǂp < 0.01, ǂǂǂp < 0.001) and to correspondingly equal concentrations of HDACis alone (▲p < 0.05, ▲▲p < 0.01, ▲▲▲p < 0.001)