Identification of NFATc1 responsive sequences in the TRAIL promoter.

<p>(<b>A</b>) Double-stranded oligonucleotides corresponding to all of the potential transcriptional binding sites in human TRAIL promoter −169 to −33 were synthesized and used for DNA binding analysis. The putative Sp1 binding sequences are boxed, which are also overlapped with other transcription factor binding sequences. (<b>B</b>) HT29 cells were treated with or without PMA/Io for 60 min and nuclear protein extracted. Double-stranded oligonucleotides (as shown in A) were radiolabeled and tested for DNA binding by EMSA. The arrows indicated the increased binding complexes. (<b>C</b>) Total RNA from HT29 cells stably transfected with control (CON) or four individual shRNAs targeting human NFATc1 (clones 33, 34, 35, 36) was extracted and RT-PCR performed for NFATc1 mRNA expression. (<b>D and E</b>) Nuclear protein (D) or total protein (E) from HT29 cells stably transfected with control (CON) or NFATc1 shRNA (clones 33, 34, 36) was extracted. EMSA was performed (D) using probe 4 and probe 5 (sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019882#pone-0019882-g006" target="_blank">Figure 6</a>). Western blotting was performed for the analysis of TRAIL expression (E); membranes were stripped and re-probed with anti-β-actin to confirm equal loading. TRAIL signals from two separate experiments were quantitated densitometrically and expressed as fold-change with respect to β-actin (Data shown as mean ± standard error of the mean).