Levels of hMTIIa in HeLa cells transfected with gold nanoparticles.

<p>Transfections were carried out with GeneJuice (Novagen). (A) Levels of hMT-IIa mRNA. HeLa cells were transfected with 5 nM unfunctionalized (−), 5 nM control (C) or 5 nM hMTIIa (MT)-specific ssDNA functionalized gold nanoparticles. Samples were treated with 12.5 µM CdCl₂. The level of hMTIIa gene expression (normalized to <i>B2M</i>) in untransfected and induced HeLa cells was defined as 100% and all other fold inductions were expressed relative to this. The error bars were calculated as 1 standard error of the mean each way. (B) Levels of hMT-IIa protein. Induced samples were treated with 12.5 µM CdCl₂ (+Cd). HeLa cells were transfected with unfunctionalized (−), control (C) or hMT-IIa (MT) ssDNA-functionalized nanoparticles. Proteins extracted from HeLa cells were analyzed using Western blots and hMT-IIa-specific antibodies. α/β tubulin was used as a loading control. Quantification of the levels of hMT-IIA protein relative to α/β tubulin is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099458#pone.0099458.s008" target="_blank">Table S2C</a>.</p

Levels of hMT-IIa in HeLa cells transfected with increasing concentrations of hMT-IIa or control ssDNA.

<p>Transfections were carried out with GeneJuice (Novagen). (A) Levels of hMT-IIa mRNA. All samples were induced samples with 12.5 µM CdCl₂. Total hMT-IIa activity relative to <i>B2M</i> in cells transfected with varying levels of ssDNA was normalized to hMT-IIa-<i>B2M</i> expression in cells transfected with 0 nM ssDNA giving us a value for hMT-IIa activity in the presence of x nM ssDNA. The levels of hMT-IIa transcript in cells transfected with control ssDNA (C) was set to 1.0. The level of hMT-IIa gene expression (MT) in cells transfected with hMT-IIa ssDNA was compared to the levels of hMT-IIa in cells transfected with control ssDNA at each concentration providing a measure of the reduction in hMT-IIa activity in hMT-IIa ssDNA transfected compared to those transfected with control ssDNA. The error bars were calculated as 1 standard error of the mean each way. (B) Levels of hMT-IIa protein. All samples were induced with 12.5 µM CdCl₂. HeLa cells were transfected with hMT-IIa-specific ssDNA (ssDNA). Proteins extracted from HeLa cells were analyzed using Western blots and hMT-IIa-specific antibodies. α/β tubulin was used as a loading control. Quantification of the levels of hMT-IIA protein relative to α/β tubulin is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099458#pone.0099458.s008" target="_blank">Table S2B</a>.</p

Fluoresence microscopy images showing localization of FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in live HeLa cells.

<p>HeLa cells were transfected with FITC-tagged hMT-IIa-specific sequence functionalized nanoparticles in the absence (NanoParticles column) or the presence of the transfection reagents Matra, Lipofectamine 2000 or GeneJuice. Control cells were treated with neither nanoparticles nor transfection reagents. The same signal intensity range was used for all FITC images. The scale bar represents 50 microns.</p

Quantification of the levels of hMT-IIa transcript in HeLa cells after treatment with ssDNA-functionalized nanoparticles.

<p>Induced samples were treated with 12.5 µM CdCl₂ (+Cd). HeLa cells were transfected with 5 nM unfunctionalized (−), 5 nM control (C) or 5 nM hMTIIa (MT)-specific ssDNA functionalized gold nanoparticles. The level of hMTIIa gene expression (normalized to <i>B2M</i>) in HeLa cells transfected with unfunctionalized (−)gold nanoparticles was normalized to 100% and all other fold inductions were expressed relative to this. The error bars were calculated as 1 standard error of the mean each way and represented as a percentage of the activity.</p

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 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

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

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

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