Analysis of induction in cadmium chloride-treated cells transfected with TFBS-UR plasmids.

<p>HEK293 cells transfected with a plasmid pool, that included the plasmids listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s003" target="_blank">Table S2</a> and pRL-SV40 and were subsequently treated with cadmium. (A) Microarray-based detection of TF derived activation of UR expression. (B) qPCR-based detection of TF-derived activation of UR expression. Values are presented as log2 treatments of the fold induction of the TFBS-directed UR expression after treatment with the inducer of interest. The grey bar represents treatment-independent changes in the system. TFBS marked with * represent treatment-dependent effects on the TF library. Numerical data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s004" target="_blank">Table S3</a>. A statistical analysis of the qPCR assay data is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone-0050521-g003" target="_blank">Figure 3</a>.</p

Induction of the TF proteins of interest in HEK293 cells after treatment with forskolin, TPA and cadmium.

<p>Proteins extracted from treated and control cells were analyzed using Western blots and TF-specific antibodies. The levels of phosphorylated TFs and inactive TFs were analyzed for (A) CREB and ATF, (B) IκB, (C) c-jun and (D) SP1. Tubulin was used as a loading control. Quantification of the levels of protein on the Western blots showed a 1.6 and 1.3 fold increase in P-CREB and P-ATF after treatment with forskolin and a 1.5 and 1.6 fold increase in P-IκB, and P-c-jun after treatment with TPA. Treatment of HEK293 cells with cadmium chloride, dexamethasone, forskolin and TPA resulted in a 1.1, 1.1. 1.0 and 1.0 fold increase in the levels of SP1 protein. (E) Increased <i>hMTIIA</i> gene expression in HEK293 cells after treatment with cadmium. Expression of the cadmium-responsive <i>hMTIIa</i> gene was normalized to the expression of the chromosomal reference gene <i>B2M.</i> Abbreviations: -, carrier only control; C, cadmium; D, dexamethasone; F, forskolin; T, TPA.</p

Induction of selected TFBS-directed UR expression in HEK293 cells after treatment with cadmium, dexamethasone, TPA and forskolin.

<p>HEK293 cells transfected with a plasmid pool, that included the plasmids listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s003" target="_blank">Table S2</a> and pRL-SV40 and were subsequently treated with drugs of interest. (A) MRE-directed UR expression after treatment with cadmium. (B) GRE-directed UR expression after treatment with dexamethasone. (C) NF-κB-directed UR expression after treatment with TPA. (D) CREB-directed UR expression after treatment with forskolin. Values are presented as log2 treatments of the fold induction of the TFBS-directed UR expression after treatment with the inducer of interest. The error bars are calculated as 1 standard error of the mean each way.</p

qPCR analysis of induction of TFBS-directed UR expression in treated cells transfected with TFBS-UR plasmids.

<p>The statistical model calculated a posterior probability distribution over the mean of the log normalized fold induction. The p-value indicated the posterior probability that there was no difference in expression levels between the control and treatment samples. 95% credible intervals were also calculated for the mean log normalized fold induction and indicate the region where there is a 95% probability that the mean effect lies within it. Bars not crossing the 0 line show significant evidence for an effect following treatment with the inducer of interest.</p

A schematic representation of the method.

<p>In each reporter plasmid, the transcription factor binding site (TFBS) and the thymidine kinase promoter (P_TK) were present upstream of the transcriptional start site (TSS) and the unique DNA reporter (UR) sequence. The cassette was flanked by two poly(A) signals to prevent transcriptional interference due to the circular plasmid. Each TFBS was assigned a specific UR sequence to act as a signature for its corresponding TF activity. These plasmids were tranfected into cells and the cells treated with compounds of interest, mRNA was isolated, reverse transcribed and analyzed on two detection platforms. For microarray analysis, cDNA was amplified by PCR using a Cy3 or Cy5-labelled universal sense forward primer (Cy3/Cy5-AG_URF) in conjunction with a universal antisense reverse primer (prMJ264) to generate a mixture of 120 bp fluorescently labelled PCR amplicons that could be analyzed on DNA microarrays. For the qPCR reaction, a forward primer, specific for each UR, was used in combination with a universal FAM-labelled hydrolysis probe (prMJ245) and a universal reverse primer (prMJ264).</p

Activation of transcription factors by specific treatments on the qPCR platform.

<p>HEK293 cells transfected with pool of plasmids (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s003" target="_blank">Table S2</a> and pRL-SV40) and were subsequently treated with chemicals of interest. Values are presented as log2 treatments of the fold induction of the TFBS-directed UR expression after treatment with the inducer of interest. The errors are calculated as 1 standard error of the mean each way. P-values indicate the posterior probability that there was no difference in expression levels between the control and treatment samples so a lower p-value would indicate a greater likelihood that there was a difference between the control and treatment samples. Abbreviations: IBMX: 3-isobutyl-1-methylxanthine, EHNA: erythro-9-(2-hydroxy-3-nonyl)adenine.</p