Journal:

Article Title: Sirt3-Mediated Deacetylation of Evolutionarily Conserved Lysine 122 Regulates MnSOD Activity in Response to Stress

doi: 10.1016/j.molcel.2010.12.013

Figure Lengend Snippet: (a) Sirt3+/+ and Sirt3−/− mice at 12 weeks were exposed to sham or 2 × 2 Gy exposure every 24 hours. Livers were harvested 20 hours post-exposure, and mitochondria were isolated. MnSOD activity in MEFs was measured via a competitive inhibition assay as described (Spitz and Oberley, 1989). Activity data for MnSOD is presented as units of SOD activity per milligram of protein. (b) Mitochondrial extracts from above were analyzed for acetylation of MnSOD lysine 122 via mass spectrometry. Results are presented as fold change from the untreated, wild-type mouse livers. (c) Sirt3−/− mouse livers exposed to IR exhibit marked cytoplasmic vacuolation of periportal to midzonal hepatocytes. The wild-type and Sirt3−/− livers without and with exposure to IR were H & E stained and scored for degree of hepatocellular cytoplasmic vacuolation; low, medium and high by a pathologist (AKO). Representative micrographs are shown. Scale bar = 80 μm. (d) Quantification of the H & E for liver cells. Liver cells were scored as low = no detectable cytoplasmic vacuolation, med = moderate dilation of the cytoplasm by clear space primarily affecting periportal hepatocytes, high = severe dilation of the cytoplasm by clear space and poorly defined clear vacuoles primarily affecting periportal to midzonal hepatocytes. (e) Apoptosis was determined in the Sirt3+/+ and Sirt3−/− livers exposed to IR. Apoptotic cells were identified in liver sections by TUNEL assay. Sections were scored by a pathologist blinded to the groupings. TUNEL-positive cells were counted in 10 randomly selected 200X fields per liver section. Results in this figure are reported as the average number of positive cells per field. Data are presented as the average +/− SD. * indicates P < 0.05 and ** indicates P < 0.01. (f) Irradiated Sirt3−/− mouse liver cells exhibit increased anti-nitrotyrosine IHC staining. Liver tissue from wild-type and Sirt3−/− mice were stained with an anti-nitrotyrosine antibody (StressMarq Biosciences Inc.). A representative micrograph is shown. Scale bar = 80 μm. See Figure S7E for quantification.

Article Snippet: Liver tissue from wild-type and Sirt3 −/− mice were stained with an anti-nitrotyrosine antibody (StressMarq Biosciences Inc.).

Techniques: Isolation, Activity Assay, Inhibition, Mass Spectrometry, Staining, TUNEL Assay, Irradiation, Immunohistochemistry

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Body Mass Index (BMI) and Its Influence on the Cardiovascular and Operative Risk Profile in Coronary Artery Bypass Grafting Patients: Impact of Inflammation and Leptin

doi: 10.1155/2020/5724024

Figure Lengend Snippet: Determination of the oxidative stress parameters 3-nitrotyrosine (3-NT) (a) and 8-Isoprostane (b) by commercial ELISA assays of a proof of increased reactive oxygen and nitrogen species formation in patients with leptin levels higher than the median. Data are mean ± SEM. (c, d) Significant correlation of leptin and levels of the serum oxidative stress marker 3-NT in 40 patients ( p values and correlation coefficients are provided in the graphs). Measured 3-NT staining was either normalized to Ponceau S staining (c) or transferrin content (d).

Article Snippet: Levels of 3-NT and 8-isoprostane, as markers of oxidative stress, were determined in human serum using a commercial ELISA kit (human immunoassay, 3-NT: #CSB-E14324h, Cusabio, Wuhan, Hubei Province 430206, China; 8-isoprostane: #MBS109360, San Diego, CA, USA) following the instructions of the vendor and previously [ ].

Techniques: Enzyme-linked Immunosorbent Assay, Marker, Staining

Journal: JVS-Vascular Science

Article Title: Zinc deficiency impairs ischemia-induced angiogenesis

doi: 10.1016/j.jvssci.2021.09.023

Figure Lengend Snippet: Zinc deficiency ( ZD ) increased the rate of oxidative damage in ischemic muscles. A, Production of reactive oxygen species (ROS) was evaluated by immunostaining with dihydroethidium (DHE) on postoperative day 28 (×400 for each field; red ). Serum levels of the derivatives of reactive oxygen metabolites (d-ROMs; B ) and nitrotyrosine (C) in the ZD mice or control wild-type (WT) mice after hindlimb surgery (n = 8 in each group). D, Messenger RNA (mRNA) levels of Nox2, p22 phox , p47 phox , and p67 phox in the ischemic muscle in the ZD mice or control WT mice on postoperative day 28. mRNA levels were measured using the real-time polymerase chain reaction (PCR) method (n = 8 in each group). All results were normalized to glyceraldehyde 3-phosphate dehydrogenase. ∗ P < .01 vs control.

Article Snippet: The serum nitrotyrosine levels were measured using a nitrotyrosine enzyme-linked immunosorbent assay kit (StressMarq Biosciences Inc, Victoria, British Columbia, Canada) according to the manufacturer's instruction.

Techniques: Immunostaining, Real-time Polymerase Chain Reaction

Journal: Europace

Article Title: GSTP1 inhibits angiotensin II-induced atrial fibrillation by regulating ferroptosis

doi: 10.1093/europace/euaf083

Figure Lengend Snippet: Overexpression of GSTP1 can improve Ang II-induced oxidative stress. ( A ) DHE fluorescence staining images (left) and their corresponding fluorescence intensity statistics (right) ( n = 5); ( B ) 3-NT immunohistochemical staining images (left) and statistics of expression levels (right) ( n = 5); ( C ) western blot analysis of NOX4 protein expression levels, a marker of oxidative stress, in mice from various groups ( n = 6); ( D ) measurement of serum MDA content in mice from different groups ( n = 5); ( E ) measurement of serum GPX activity in mice from various groups ( n = 5). Statistical analysis was performed with one-way ANOVA ( A–E ). A significance level of * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 was considered statistically significant.

Article Snippet: Apply the endogenous peroxidase blocker for 10 min. Block with 5% Bovine Serum Albumin (BSA) and let it sit for 30 min. Add primary antibodies, including CD68 (1:100, ARG10514 , arigobio), 3-Nitrotyrosine (3-NT) (1:100, bs-8551R, bioss), and α-SMA (1:500, ARG66381 , arigo), and incubate overnight at 4°C.

Techniques: Over Expression, Fluorescence, Staining, Immunohistochemical staining, Expressing, Western Blot, Marker, Activity Assay