Supplemental Results

1. A Supplemental Results Online Figure S1 B Online Figure S1. Norepinephrine increases ROS production. (A): H9c2 cells were treated with 10 M norepinephrine and intracellular ROS levels were measured with DCF-DA at different time points (4, 10, 16, 24, 36 and 48 hours). (B) H9c2 cells were treated with norepinephrine (NE) for 16 hours in the absence or presence of prazosin (1 M). ROS were measured with DCF-DA. Data are mean  SEM, n = 6. * P < 0.05 vs. control. 1

2. Online Figure S2 A B Online Figure S2. Norepinephrine increases ROS production. H9c2 cells and isolated intact fetal hearts (ex vivo) were treated with 10 M norepinephrine (NE) in the absence or presence of N-acetylcysterine (NAC, 1 mM), apocynin (Apo, 0.5 mM) or diphenyleneiodonium (DPI, 10 μM) for 16 hours. ROS were measured with the quantification of confocal images of dihydroethidium (DHE) fluorescence. (A): H9c2 cells. (B): ex vivo fetal hearts. Data are mean  SEM, n = 8. * P < 0.05 vs. control. 2

3. Online Figure S3 Online Figure S3. Effect of norepinephrine on Egr-1 and Sp1 protein abundance. H9c2 cells were treated with 10 M norepinephrine (NE) for 48 hours. Cytosol and nuclear protein abundance of Egr-1 and Sp1 was determined with Western blot. Data are mean  SEM, n = 4. 3

4. H9c2 aorta Protein: 40 60 80 40 g Nox2 Fetalhearts aorta Nox1 Nox2 Nox4 -actin actin Control NE Nox2 actin Online Figure S4 A B C Online Figure S4. Expression of Nox1, Nox2 and Nox4 in fetal hearts and H9c2 cells. (A): Nox2 protein expression was readily detected in rat aortic smooth muscle but not in H9c2 cells. (B): mRNA of Nox1 and Nox4, but not Nox2, was detected in H9c2 cells. (C): Nox2 protein expression was detected in rat aortic smooth muscle but not in isolated fetal rat hearts in the absence or presence of 10 M norepinephrine (NE) for 48 hours. 4