Nged phosphorylation at Ser1177 (Fig S4B) upon Sirt3 knockdown. Having said that, we observed a decreased phosphorylation of Thr495 following transient knockdown of Sirt3 (Fig S4C), equivalent to an increased enzymatic activity. With each other with an increased coupling of eNOS monomers (Fig S4D) this may very well be a compensatory effect secondary to an improved mitochondrial ROS accumulation upon Sirt3 deficiency. To assess the functional relevance of improved eNOS activity, nitric oxide (NO) generation was assessed in presence or absence of L-NIO, an unselective nitric oxide synthase inhibitor, employing DAF-2 diacetate. L-NIO effectively lowered NO generation, having said that, no significant distinction was observed upon transient knockdown of Sirt3 compared with controls, neither in presence nor in absence of L-NIO (Fig S4E). Hence, increased eNOS activity upon Sirt3 deficiency will not generate a detectable rise in NO. Nonetheless, increased eNOS coupling could contribute to counteract elevated ROS levels upon Sirt3 deficiency.Page six ofBasic Res Cardiol (2016) 111:Relaxation [ precontraction]AUC [Arbitrary units]Fig. two Endothelial function is mildly impaired in Sirt3-/mice within a superoxide-dependent manner. a Relaxation of aortic rings in response to escalating doses of acetylcholine (ACh) in Sirt3-/- compared with wildtype mice fed a common chow. b As within a following 12 weeks of a high-cholesterol diet. c As in b, in presence of an excess of pegylated superoxide dismutase (PEG-SOD, 220 U/ml), scavenging superoxide. d As in b, following preincubation with L-NAME (0.three mM, 30 min). n = five mice per group, four rings per mouse, quantification from the locations below the curve (AUC), boxplots show interquartile ranges, whiskers indicate minima and maxima(A)Relaxation [ precontraction]-20 0 20 40 60Relaxation to acetylcholine (ACh)AUC [Arbitrary units]normal diet900 800 700 600 50Area under the curve (AUC)p=0.wildtype Sirt3 k.o.1001E 3E 09 1E 09 -0 3E eight 1E 08 3E 07 -0 1E 7 3E 06 1E 06 -0 3E 5 1E 05 -0w ild ty peACh [M](B)-20 0 20 40 60 80 100Relaxation to acetylcholine (ACh)high-cholesterol diet900 800 700 600 50Area under the curve (AUC)p=0.581063-34-5 manufacturer wildtype Sirt3 k.o.w ild ty pe1E 3E 09 1E 09 -0 3E eight 1E 08 3E 07 -0 1E 7 3E 06 1E 06 3E 05 1E 05 -0ACh [M](C)Relaxation [ precontraction]-20 0 20 40 60 80 100Relaxation to acetylcholine (ACh) following PEG-SOD (220U/ml)AUC [Arbitrary units]high-cholesterol diet1100 1000 900 800 700 600 50Area beneath the curve (AUC)p=0.6-Hydroxybenzo[d]thiazole-2-carbonitrile site wildtype Sirt3 k.PMID:23724934 o.Si rtp=0.w ild ty pe1E 3E 09 1E 09 3E 08 -0 1E eight 3E 07 1E 07 -0 3E 6 1E 06 3E 05 -0 1E 5 -0ACh [M](D)Relaxation [ precontraction]-Relaxation to acetylcholine (ACh) following L-NAME (0.3 mM)AUC [arbitrary units]wildtype Sirt3 k.o.800 600 400 200Area beneath the curve-60 -40 -20 0 20 40high-cholesterol dietSi rtSi rt3 k. o.ACh [M]w ild ty pe1E -0 3E 9 -0 1E 9 -0 3E 8 -0 1E eight -0 3E 7 -0 1E 7 -0 3E 6 -0 1E 6 -0 3E 5 -0 1E five -0k. o.k. o.Si rtk. o.Fundamental Res Cardiol (2016) 111:33 Fig. 3 Loss of Sirt3 decreases endothelial SOD2 activity and increases SOD2 expression devoid of affecting other ROSscavenging or creating systems. a Superoxide dismutase two (SOD2) activity based on superoxide dismutating capacity in HAEC following siRNA-mediated knockdown of Sirt3; enzymatic activity was normalized to SOD2 protein expression; medians and single information points are shown. b Overall SOD2 activity, as within a without having normalizing to protein content. c SOD2 mRNA (left) and protein (proper) expression in HAEC following siRNAmediated knockd.