Journal: Oxidative Medicine and Cellular Longevity

Article Title: In Situ Evaluation of Oxidative Stress in Rat Fatty Liver Induced by a Methionine- and Choline-Deficient Diet

doi: 10.1155/2016/9307064

Figure Lengend Snippet: Liver of rats submitted to control diet for 2 weeks. (a) Representative photomicrographs of PAS reaction for glycogen, abundant in all hepatocytes. (b) Nile Red-induced fluorescence of small neutral lipid droplets in the perisinusoidal cytoplasm of hepatocytes. (c) Diaminobenzidine-Mn 2+ -Co 2+ reaction for Reactive Oxygen Species (ROS); intense reaction in periportal hepatocytes and occasional Kupffer cells in the midzone or pericentral regions. (d) Immunoreaction against dinitrophenyl (DNP) groups for demonstrating carbonyl groups derivatized with dinitrophenyl hydrazine (DNPH); these are present in perisinusoidal and canalicular membrane domains of hepatocytes. (e) Immunoreaction for visualizing the expression of Mn-dependent Superoxide Dismutase 2 (SOD2); moderately intense staining in the cytoplasm of periportal and pericentral hepatocytes. P: branch of portal vein; CL: branch of centrolobular vein. Scale bar: 50 μ m (insets in (d) and (e) represent image details at higher magnification).

Article Snippet: Endogenous peroxidase activity blocking was performed with 10% methanol-3% H 2 O 2 in PBS for 20 min. After washing twice for 5 min in PBS, nonspecific site blocking was performed with 2.5% normal horse serum in PBS for 1 h. The sections were then incubated with a primary rabbit polyclonal antibody against SOD2 (NB100-1992; Novus Biologicals, Littleton, CO 80120, USA) diluted 1 : 300 in 2.5% normal horse serum at room temperature for 1 h. After two further 5 min washing instances in PBS, the sections were incubated with ImmPRESS HRP Universal Antibody (anti-mouse Ig/anti-rabbit Ig, peroxidase) Polymer Detection Kit (MP-7500; Vector Laboratories, Burlingame, CA 94010, USA) for 30 min at room temperature.

Techniques: Control, Fluorescence, Membrane, Expressing, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: In Situ Evaluation of Oxidative Stress in Rat Fatty Liver Induced by a Methionine- and Choline-Deficient Diet

doi: 10.1155/2016/9307064

Figure Lengend Snippet: Expression of Mn-dependent Superoxide Dismutase 2 (SOD2) in the liver of rats fed with the MCD diet for 1–4 weeks. Representative photomicrographs. Immunoreactivity is concentrated in the thin rim of cytoplasm surrounding the large lipid droplets. P: branch of portal vein; CL: branch of centrolobular vein. Scale bar: 50 μ m.

Article Snippet: Endogenous peroxidase activity blocking was performed with 10% methanol-3% H 2 O 2 in PBS for 20 min. After washing twice for 5 min in PBS, nonspecific site blocking was performed with 2.5% normal horse serum in PBS for 1 h. The sections were then incubated with a primary rabbit polyclonal antibody against SOD2 (NB100-1992; Novus Biologicals, Littleton, CO 80120, USA) diluted 1 : 300 in 2.5% normal horse serum at room temperature for 1 h. After two further 5 min washing instances in PBS, the sections were incubated with ImmPRESS HRP Universal Antibody (anti-mouse Ig/anti-rabbit Ig, peroxidase) Polymer Detection Kit (MP-7500; Vector Laboratories, Burlingame, CA 94010, USA) for 30 min at room temperature.

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: In Situ Evaluation of Oxidative Stress in Rat Fatty Liver Induced by a Methionine- and Choline-Deficient Diet

doi: 10.1155/2016/9307064

Figure Lengend Snippet: Evaluation of SOD2 expression in livers of the rats fed with control or MCD diet for 1–4 weeks. (a) Comparison of SOD2 expression in the liver of rats fed with control and MCD diet for 1–4 weeks. Histograms representing the ratio between optical density (OD) values of homogenates of the liver of rats fed with the MCD diet for 1 to 4 weeks and the OD of control livers (b).

Article Snippet: Endogenous peroxidase activity blocking was performed with 10% methanol-3% H 2 O 2 in PBS for 20 min. After washing twice for 5 min in PBS, nonspecific site blocking was performed with 2.5% normal horse serum in PBS for 1 h. The sections were then incubated with a primary rabbit polyclonal antibody against SOD2 (NB100-1992; Novus Biologicals, Littleton, CO 80120, USA) diluted 1 : 300 in 2.5% normal horse serum at room temperature for 1 h. After two further 5 min washing instances in PBS, the sections were incubated with ImmPRESS HRP Universal Antibody (anti-mouse Ig/anti-rabbit Ig, peroxidase) Polymer Detection Kit (MP-7500; Vector Laboratories, Burlingame, CA 94010, USA) for 30 min at room temperature.

Techniques: Expressing, Control, Comparison

Journal: Cell Death & Disease

Article Title: PINK1-parkin-mediated neuronal mitophagy deficiency in prion disease

doi: 10.1038/s41419-022-04613-2

Figure Lengend Snippet: A , B Western blots of mitochondrial protein (TOMM40, COXIV, SOD2) expressions in N2a cells with A overexpressed and B knocked down (siRNA) parkin cells, with and without PrP106-126, nicotinamide mononucleotide (NMN), and urolithin A (UA) treatments (Tubulin and β-actin served as loading controls.) C , D Comparisons of mitochondrial protein levels, relative to controls, in cells from A and B , respectively. Data were mean (SD). ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. All experiments were repeated at least three times.

Article Snippet: The following primary antibodies were used for blotting: anti-parkin antibody (Abcam, catalog no. ab77924; Santa Cruz Biotechnology, catalog no. sc-32282), PINK1 antibody (Novus Biologicals, catalog no. BC100–494), caspase 3 polyclonal antibody (Proteintech, catalog no. 19677-1-AP), anti-optineurin antibody [EPR20654] (Abcam, catalog no. ab213556), phospho-TBK1/NAK (Ser172) (D52C2) XP rabbit monoclonal antibody (Cell Signaling Technology, catalog no. 5483), LC3 rabbit polyclonal antibody (Proteintech, catalog no. 14600-1-AP), TOMM40 rabbit polyclonal antibody (Proteintech, catalog no. 18409-1-AP), SOD2 rabbit polyclonal antibody (Proteintech, catalog no. 24127-1-AP), COXIV rabbit polyclonal antibody (Proteintech, catalog no. 11242-1-AP), GAPDH monoclonal antibody (Proteintech, catalog no. 60004-1-Ig), beta-actin monoclonal antibody (Proteintech, catalog no. 66009-1-Ig), and alpha-tubulin polyclonal antibody (Proteintech, catalog no. 11224-1-AP).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: A Preliminary Study of the Effect of Quercetin on Cytotoxicity, Apoptosis, and Stress Responses in Glioblastoma Cell Lines

doi: 10.3390/ijms23031345

Figure Lengend Snippet: The effect of QCT on oxidative stress in glioblastoma cells. A172 and LBC3 cells were incubated with 50 or 100 μmol/L of QCT for 24 h or 48 h. ROS generation in A172 ( A ) and LBC3 ( B ) cells. RT-qPCR analysis of antioxidant-enzyme genes Sod1 and Sod2 in A172 ( C ) and LBC3 ( D ) cell lines. Results shown as relative fold change in mRNA expression in comparison to untreated controls, where expression level was set as 1. Western blot analysis of SOD1 and SOD2 expressions in A172 and LBC3 cells ( E ). Mean ± SD from three independent experiments are shown. * p < 0.05.

Article Snippet: HRP-conjugated anti-rabbit IgG, polyclonal (rabbit) anti-β-tubulin, monoclonal (rabbit) anti-CHOP, monoclonal (rabbit) anti-Casp 3 (cleaved), monoclonal (rabbit) anti-Casp 9 (cleaved), polyclonal (rabbit) anti-SOD1, and monoclonal (rabbit) anti-SOD2 antibodies were provided by Cell Signaling Technology (Boston, MA, USA).

Techniques: Incubation, Quantitative RT-PCR, Expressing, Comparison, Western Blot

Journal: The American Journal of Pathology

Article Title: Hepatitis C and Alcohol Exacerbate Liver Injury by Suppression of FOXO3

doi: 10.1016/j.ajpath.2013.08.013

Figure Lengend Snippet: Effects of HCV and alcohol on FOXO3 transcriptional activity. A: Real-time RT-PCR for FOXO3-dependent genes, FOXO3, SOD2, Bim, and GADD45, and a control, non-FOXO target gene, SOD1, in RNA isolated from control (black bars) and 5-day HCV-infected (gray bars) Huh7.5 cells. n = 6 to 18 individual RNA preparations. P values by Wilcoxon signed-rank test for the comparison of infected and noninfected conditions were as follows: FOXO3, P < 0.001; SOD2, P < 0.001; BIM, P < 0.005; GADD45, P < 0.05; SOD1, P = 0.86. B: Time course of mRNA expression changes determined by real-time PCR for FOXO3 and its target genes, Bim and SOD2, in control (black bars) and HCV-infected (gray bars) cells treated with 50 mmol/L ethanol for the indicated times. N = 3 to 6 individual preparations. C: HCV-infected and noninfected Huh7.5 cells were incubated with 50 mmol/L ethanol for 48 hours. Western blots for FOXO3, SOD2, Bim, and β-actin (loading control) are presented for whole cell lysates prepared at different times of alcohol exposure. Similar results were obtained in four independent experiments. D: SOD2 protein levels were measured by densitometry analysis of immunoblots performed as in A. n = 4. E: Kinetics of SOD2 degradation after cycloheximide treatment, determined as in D (n = 3 experiments). F: pMiR-Target-Luciferase-SOD2 3′-UTR reporter expression in HCV-infected and noninfected Huh7.5 cells incubated where indicated with 50 mmol/L ethanol for 48 hours. n = 3. ∗P < 0.05, ∗∗P < 0.01.

Article Snippet: Primary antibodies used were anti-FOXO3 (75D8), anti–phospho-FOXO3 (Ser253), anti-pan Akt (11E7), anti–phospho-Akt (Ser473), anti-Bim, and rabbit anti-caspase 3 (D175) from Cell Signaling, anti-SOD2 (sc-30080), goat anti–intercellular adhesion molecule (ICAM) 1, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA), anti-HCV core from Affinity Bioreagents (Golden, CO), and rabbit anti-SOD2 and mouse anti–β-actin from Sigma-Aldrich.

Techniques: Activity Assay, Quantitative RT-PCR, Isolation, Infection, Expressing, Real-time Polymerase Chain Reaction, Incubation, Western Blot, Luciferase

Journal: The American Journal of Pathology

Article Title: Hepatitis C and Alcohol Exacerbate Liver Injury by Suppression of FOXO3

doi: 10.1016/j.ajpath.2013.08.013

Figure Lengend Snippet: Effects of alcohol feeding on mouse liver. Mice from the four genotypes were fed either a Lieber-DeCarli liquid alcohol diet or a control, alcohol-free liquid diet for 3 weeks, as described in Materials and Methods. A: Serum ALT values expressed for each mouse were expressed as fold elevation compared with WT mouse on the control diet. ALT in the ethanol-fed HCV/Sod2+/− group was significantly different from the other groups by analysis of variance (P < 0.001). White squares, pair fed; red circles, alcohol fed. B: Representative H&E-stained liver histopathological characteristics for the different groups. C: Western blot analysis for inflammation and apoptosis markers in 3-week alcohol-fed and control-fed mice. D: Higher magnification of features of the HCV/Sod2+/− alcohol-treated livers demonstrating ballooning degeneration (top panel) and lobular inflammation (bottom panel). Features are indicated by arrows. Casp, caspase.

Article Snippet: Primary antibodies used were anti-FOXO3 (75D8), anti–phospho-FOXO3 (Ser253), anti-pan Akt (11E7), anti–phospho-Akt (Ser473), anti-Bim, and rabbit anti-caspase 3 (D175) from Cell Signaling, anti-SOD2 (sc-30080), goat anti–intercellular adhesion molecule (ICAM) 1, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA), anti-HCV core from Affinity Bioreagents (Golden, CO), and rabbit anti-SOD2 and mouse anti–β-actin from Sigma-Aldrich.

Techniques: Staining, Western Blot

Journal: The American Journal of Pathology

Article Title: Hepatitis C and Alcohol Exacerbate Liver Injury by Suppression of FOXO3

doi: 10.1016/j.ajpath.2013.08.013

Figure Lengend Snippet: SOD2 and FOXO3 expression and distribution in alcohol-fed mouse livers. A: Correlation of ALT with postalcohol SOD2 level in liver. SOD2 expression relative to β-actin was determined by densitometry of Western blots and plotted versus serum ALT. Blue diamonds, control-fed; red circles, alcohol fed. B: Comparison of the effects of alcohol feeding on SOD2 protein abundance in liver for the four genotypes. Liver homogenates were analyzed by using Western blot analysis after 3 weeks of alcohol or control diet feeding. Each lane represents homogenate from a separate mouse. C: FOXO3 immunohistological characteristics are presented. For each genotype, paraffin sections of liver were stained for FOXO3; and for each diet, images are shown at low power (10× objective, left panels) and at higher magnification (right panels), as indicated. The arrow indicates a cell with cytosolic immunoreactivity for FOXO3. D: Quantitative analysis of proportion of hepatocytes displaying cytosolic FOXO3 staining. Black bars, control diet; gray bars, ethanol diet. N = 3 different mouse livers for each condition. ∗∗P < 0.01 compared with Sod2+/−.

Article Snippet: Primary antibodies used were anti-FOXO3 (75D8), anti–phospho-FOXO3 (Ser253), anti-pan Akt (11E7), anti–phospho-Akt (Ser473), anti-Bim, and rabbit anti-caspase 3 (D175) from Cell Signaling, anti-SOD2 (sc-30080), goat anti–intercellular adhesion molecule (ICAM) 1, and β-actin from Santa Cruz Biotechnology (Santa Cruz, CA), anti-HCV core from Affinity Bioreagents (Golden, CO), and rabbit anti-SOD2 and mouse anti–β-actin from Sigma-Aldrich.

Techniques: Expressing, Western Blot, Staining