Figure 6. ROS levels in PDGF stimulated SK-N-BE cells.

(A) Cells were incubated 18h in medium containing 0.2% FBS, loaded with 10µM DCHF-DA in the presence or absence of the intracellular calcium chelator, BAPTA-AM (10µM), and then stimulated with 15ng/ml of PDGF as described in Materials and Methods. ROS levels were measured fluorimetrically at the time intervals indicated. Ca⁺⁺-independent ROS were measured in presence of BAPTA-AM. Ca⁺⁺-dependent ROS levels were derived from the assays performed in the presence or absence of BAPTA-AM. Total levels of ROS induced by PDGF were also measured in the absence (Total) or presence of the NADPH oxidase inhibitor AEBSF. Values are Mean +/- SEM of three independent experiments performed in triplicate. * p< 0.01 and ** p< 0.05 vs not stimulated; § p<0.01 vs the corresponding time point of Total (Ctr) curve. (B) Cells were transfected by electroporation with siRNA to p22^phox (siRNA p22^phox) or control, scrambled siRNA (scramble) as described in Materials and Methods. 24h after transfection cells were incubated in medium containing 0.2%FBS for 18h and then stimulated with 15ng/ml of PDGF for 15min. An aliquot of cell medium was collected and analyzed for H₂O₂ levels as described in Materials and Methods. The histograms show the mean +/- SEM values of three independent experiments. * p< 0.01 vs Ctr. ** p< 0.01 vs PDGF stimulated scramble.