Promoter activities of pGL4.10-<i>ek1</i>(-1966/+1) construct in HepG2, HCT116 and MCF-7 cells.

<p>Each bar represents the mean ± SEM of triplicate samples from three independent experiments. (** <i>p</i> < 0.01; ***<i>p</i> < 0.001 vs. promoterless pGL4.10[<i>luc2</i>]).</p

Effect of TSA on the activities of wild type and Sp(-40/-31) mutated <i>ek1</i> minimal promoters.

<p>A. Promoter activity of pGL4.10-<i>ek1</i>(-69/+1) reporter construct in HCT116 cells treated with the indicated concentrations of TSA for 24 hours (*<i>p</i> < 0.001; **<i>p</i> < 0.01; ***<i>p</i> < 0.05 vs. DMSO control; #<i>p</i> < 0.05, significant within TSA treatment group). B. Promoter activity of pGL4.10-<i>ek1</i>(-69/+1) reporter construct in HCT116 cells treated with 1 μM of TSA for the indicated time points (*<i>p</i> < 0.05 vs. DMSO control). C. Activities of wild type <i>ek1</i> minimal promoter and Sp(-40/-31)-mutated <i>ek1</i> minimal promoter after treatment with 1 μM of TSA for 24 hours. Each bar represents the mean ± SEM of triplicate samples from three independent experiments. D. Effect of TSA on <i>ek1</i> gene expression in HCT116 cells. HCT116 cells were treated with 1 μM of TSA for 24 hours (**<i>p</i> < 0.01 vs. DMSO control). Each bar represents the mean ± SEM of triplicate samples from three independent experiments.</p

Primers used for generating promoter-luciferase constructs and PCR site-directed mutagenesis.

<p>Primers used for generating promoter-luciferase constructs and PCR site-directed mutagenesis.</p

Effects TSA treatment on the binding of Sp1, Sp3 and RNA polymerase II to the <i>ek1</i> minimal promoter region.

<p>ChIP analysis was performed to confirm the interaction of (A) Sp proteins and (B) RNA polymerase II with the promoter under 1 μM TSA treatment for 24 hours. PCR amplification products were resolved on 2% (w/v) agarose gel and visualized by EtBr staining. Band intensities were quantitated with Image J 1.42 and the relative intensities (compared to negative control) of PCR products from Sp1 and Sp3 immunoprecipitates were plotted. Each bar represents standard error of means (SEM) from two independent experiments. M: GeneRuler^™ DNA Ladder Mix; T: total input sample (unprocessed chromatin); P: positive control (amplified using GAPDH primers) and N: pre-immune normal rabbit IgG (negative control).</p

Effect of TSA on the activity of wild type <i>ek1</i> minimal promoter in HepG2 and HCT116 cells.

<p>Cells were treated with 1 μM of TSA for 24 hours (**<i>p</i> < 0.01 vs. DMSO control). Each bar represents the mean ± SEM of triplicate samples from three independent experiments.</p

ChIP analysis of <i>ek1</i> minimal promoter region for the binding of Sp1 and Sp3 in HCT116 and HepG2 cells.

<p>PCR amplification products were resolved on 2% (w/v) agarose gel and visualized by EtBr staining. Band intensities were quantitated with Image J 1.42 and the relative intensities (compared to negative control) of PCR products from Sp1 and Sp3 immunoprecipitates were plotted. Each bar represents standard error of means (SEM) from two independent experiments. M: GeneRuler^™ DNA Ladder Mix; T: total input sample (unprocessed chromatin); P: positive control (amplified using GAPDH Primers) and N: pre-immune normal rabbit IgG (negative control).</p

Effect of PKC inhibitors (PKC412 and Go 6983) on the PMA-mediated repression of <i>ckβ</i> promoter activity.

<p>(A) MCF-7 cells were individually or co-treated with 20 ng/mL PMA, 1 mM PKC412 and 0.1 mM Go 6983 for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*<i>p</i><0.05 and **<i>p</i><0.01 vs. DMSO control). (B) MCF-7 cells were individually or co-treated with 20 ng/mL PMA, 10 µM PKCε inhibitor peptide and 10 µM PKCη pseudo-substrate inhibitor for 6 hr. Each bar represents the mean ±SEM of triplicate samples from three independent experiments. (*<i>p</i><0.05 and **<i>p</i><0.01 vs. DMSO control, #<i>p</i><0.05 between the two indicated treatments).</p

Effect of PMA on the binding of Ets and GATA to the <i>ckβ</i> distal promoter.

<p>EMSA was performed using nuclear extracts from MCF-7 cells treated with or without 20 ng/mL PMA. The blot is representative of three independent experiments that produced similar results.</p

Primers used for cloning of promoter-luciferase constructs, PCR-based site-directed mutagenesis, real-time PCR and ChIP.

<p>The mutations introduced into the binding sites are italicized and in lowercase.</p><p>Primers used for cloning of promoter-luciferase constructs, PCR-based site-directed mutagenesis, real-time PCR and ChIP.</p

Sequence analysis of the <i>ckβ</i> 5′ flanking region.

<p>The predicted transcription factor binding sites are underlined. The translation start codon (ATG) is indicated in boldface.</p