Silencing of p22<i>^phox</i> inhibits the induction of DUOX 1 and 2 protein and mRNA levels by PDGF.

<p>(<b>A</b>) Identification of NOX expressed in neuroblastoma (SK-N-BE) and colon carcinoma (Caco-2) cells. Total RNA was extracted with Trizol, reverse transcribed and analyzed by PCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034405#s2" target="_blank">Materials and Methods</a>) with specific primers to NOX1, NOX2 and NOX4 (left) or NOX3 and NOX5 (right). The number of cycles was 35. (<b>B</b>) Cells were transfected by electroporation with two different (45 and 46) siRNA to p22<i>^phox</i> (siRNA p22<i>^phox</i>) or control, scrambled siRNA (scramble) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034405#s2" target="_blank">Materials and Methods</a>. 48h after transfection, total proteins were extracted and subjected to immunoblot analysis of p22<i>^phox</i>. The histograms show the mean +/- SEM values relative to scramble obtained by densitometric analysis of p22<i>^phox</i> bands normalized for α-tubulin of three independent experiments. *p< 0.01 vs scramble. (<b>C</b>) 24h after transfection cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. Total proteins were extracted and subjected to immunoblot analysis of DUOX. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) obtained by densitometric analysis of DUOX bands normalized for α-tubulin of three independent experiments. * p< 0.01 vs Ctr. (<b>D, E</b>) 24h after transfection with a mix of the two p22<i>^phox</i> siRNA, cells were incubated in medium containing 0.2% FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min, mRNA was extracted and DUOX1 and DUOX2 mRNA levels were analyzed by RT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034405#s2" target="_blank">Materials and Methods</a>. The histograms show the mean +/- SEM values relative to samples not stimulated with PDGF (Ctr) of three independent experiments. * p<0.05 vs Ctr; ** p<0.05 vs PDGF stimulated scramble.