Fig. 4.

Effect of HDACis ± HY-PDT on CDKN1A expression. a mRNA (by qRT-PCR) and b protein levels (by Western blotting) levels 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, b) or 4 h (b), as indicated. 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. CDKN1A mRNA levels were normalized to relative to those of the housekeeping gene, GAPDH. b CDKN1A protein levels were quantified by densitometry, normalized relative to the loading control (beta-actin), and expressed as folds of the reference Ct. Ct protein levels were undetectable, so their values were set as 1.0 for reference purposes. 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)