Journal: Antioxidants & Redox Signaling

Article Title: Disruption of Endothelial Cell Mitochondrial Bioenergetics in Lambs with Increased Pulmonary Blood Flow

doi: 10.1089/ars.2012.4806

Figure Lengend Snippet: l-Arginine attenuates the ADMA-mediated eNOS mitochondrial translocation and mitochondrial dysfunction in PAEC. Cells were transfected with eNOS-GFP. The mitochondria of these cells were then labeled with MitoTracker (red), and the cells were exposed to ADMA (5 μM) in the presence or absence of l-arginine (75 μM). The extent of mitochondrial localization of eNOS was then determined by measuring the intensity of yellow fluorescence (overlap of red fluorescence of MitoTracker and green fluorescence of eNOS-GFP). There is a time-dependent increase in yellow fluorescence in the ADMA treated cells, and this is blocked by the addition of l-arginine (A). Mitochondrial fractions were also isolated, and the level of eNOS protein accumulation in mitochondria was determined by Western blotting (B). ADMA increases eNOS accumulation in the mitochondria, and this is blocked by l-arginine (B). Loading was normalized by reprobing with the mitochondrial protein, VDAC and reprobed with antibodies raised against NaKATPase, Calnexin, 58K, or laminB1 to demonstrate no cross-contamination with the plasma membrane, ER membrane, Golgi, or nuclear fractions, respectively (B). A separate gel was run using a PAEC cell lysate (20 μg) to demonstrate that each antibody recognizes the ovine protein (B). ADMA also reduces the mitochondrial membrane potential as estimated by the increase in the green, momomeric form and the decrease in the aggregated red form of the DePsipher compound (C, D) Western blot analysis also revealed that ADMA decreased the levels of SOD2 (E) and increased the levels of UCP2 (F). l-Arginine treatment prevented these changes (E, F). The NOS inhibitors, l-NMMA (100 μM) and l-NAME (100 μM) also increased PAEC protein nitration (G) and reduced the mitochondrial membrane potential (H, I). The translocation of eNOS to the mitochondria was associated with NOS uncoupling as shown by an increase in NOS-derived superoxide (J) as well as an increase in mitochondrial peroxynitrite levels (K). Values are means±SEM; n=4–8. *p<0.05 versus untreated, †p<0.05 versus ADMA alone. 3-NT, 3-nitrotyrosine; ADMA, asymmetric dimethylarginine; eNOS, endothelial nitric oxide synthase; ER, endoplasmic reticulum; GFP, green fluorescent protein; l-NAME, Nω-nitro-l-arginine methyl ester hydrochloride; l-NMMA, NG-methyl-L-arginine acetate; NOS, nitric oxide synthase; PAEC, pulmonary arterial endothelial cells; SEM, standard error of the mean; SOD, superoxide dismutase; UCP2, uncoupling protein 2; VDAC, voltage-dependent anion channel. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

Article Snippet: SOD2 activity was then measured using an SOD activity kit (Enzo Life Sciences, cat #ADI-900-157).

Techniques: Translocation Assay, Transfection, Labeling, Fluorescence, Isolation, Western Blot, Nitration, Derivative Assay

Journal: Journal of Translational Medicine

Article Title: Cathepsin A inhibition attenuates myocardial infarction-induced heart failure on the functional and proteomic levels

doi: 10.1186/s12967-016-0907-8

Figure Lengend Snippet: Effect of cathepsin A inhibition in an in vitro model of ischemia. Cells were incubated for 24 h with 10 μM SAR1 or DMSO as a solvent-only control in simulated ischemia culture conditions (hypoxia and serum deprivation, see Methods section for details). a Quantitative analysis of caspase-3 activity in the total cell lysate of cells treated with SAR1 in standard conditions (normoxia; DMEM supplemented with FCS) and simulated ischemia (hypoxia; DMEM serum-free). b Cells in simulated ischemia were stained with YO-PRO-1 or PI and evaluated by flow cytometry. The percentage of cells positive to YO-PRO-1 or to PI was calculated. Results are expressed as mean ± SEM. Differences significant at p < 0.05 are marked with an asterisk (*)

Article Snippet: Caspase-3 activity was determined using a caspase-3 activity assay kit (Enzo Life Sciences, Lörrach, Germany, Cat.No.

Techniques: Inhibition, In Vitro, Incubation, Activity Assay, Staining, Flow Cytometry