LBH589 treatment enhances Bim expression of the human Karpas707 and OPM-2 myeloma cells. (A) Bim mRNA expression in Karpas707 cells after treatment with LBH589, DAC, and LBH589/DAC. Cells were treated with 15nM LBH589 for 24 hours, 500nM DAC for 72 hours, and a combination of both. The Assays on Demand detected an mRNA sequence identifying only the BimEL isoform. The mean value ± SD of 3 independent experiments is given (*P < .05). (B-C) Bim expression in the Karpas707 (B) and OPM-2 (C) cells after treatment with LBH589, DAC, and LBH589/DAC as shown by Western blot analysis. Cells were treated with 15nM LBH589 for 24 hours, 500nM DAC for 72 hours, and a combination of both. Equivalent amounts of lysates were immunoblotted with anti-Bim and anti-actin to confirm equal loading. One experiment representative of 3 is shown. (D-E) Acetylation status of histone 3 (H3) and H4 in the Karpas707 (D) and OPM-2 (E) cells after LBH589, DAC, and combination treatment. Equivalent amounts of lysates were immunoblotted with anti–acetyl H3, anti–acetyl H4, and anti-actin to confirm equal loading. One experiment representative of 3 is shown. (F) ChIP analysis of the Bim promoter in the Karpas707 cells after treatment with LBH589 and/or DAC. Chromatin DNA was immunoprecipitated with antibodies for normal IgG (negative control), anti–acetyl-histone H3 lysine 9 (Ac H3K9), anti–trimethyl-histone H3 lysine 4 (3M H3K4), anti–dimethyl-histone H3 lysine 9 (2M H3K9), and anti–histone H3 (positive control). A DNA fragment corresponding to the Bim promoter region was amplified by quantitative RT-PCR. Each ChIP and RT-PCR were repeated, respectively, 3 and 2 times (*P < .05). SDs refer to the 3 independent experiments.