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: Oxidative Medicine and Cellular Longevity

Article Title: Curcumin Ameliorates Age-Induced Tight Junction Impaired in Porcine Sertoli Cells by Inactivating the NLRP3 Inflammasome through the AMPK/SIRT3/SOD2/mtROS Signaling Pathway

doi: 10.1155/2023/1708251

Figure Lengend Snippet: Curcumin inhibits D-gal-induced decreased barrier function via inhibiting ROS and mtROS accumulation in SCs in vitro . (a) SCs undergo 48 h of treatment with D-gal (40 g/L), compound C (10 μ M), 3-TYP (50 μ M), and/or Curcumin (10 μ M). Western blotting is performed for SOD2 proteins. (b) The mtROS levels are measured using MitoSOX™ Red. (c) Total ROS levels are detected with DCFH-DA (scale bar = 50 μ m). (d) The mtROS levels are quantified by virtue of a fluorescence spectrometer (scale bar = 50 μ m). (e) SCs are treated with D-gal (40 g/L) and/or mito-TEMPO (50 μ M) for 48 h. ZO-1, Occludin, and Claudin-11 proteins are probed into via Western blotting, for which β -actin is determined as the loading control. The values are expressed in the format of mean ± SEM; ∗∗ P < 0.01 vs. the control group; ## P < 0.01 vs. the D-gal treatment group.

Article Snippet: After washing in ice-cold PBS, the acquired tissues and cells were processed with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer complemented with phenylmethylsulfonyl fluoride (PMSF, at 1 mM), followed by Western blotting in accordance with the previously mentioned methods [ ], with Occludin (diluted at 1 : 1000; bs-10011R; Bioss), ZO-1 (dilution rate 1 : 1000; bs-1329R; Bioss), p-AMPK (Ser485) (diluted at 1 : 1000; #2537; Cell Signaling Technology; Danvers; USA), Claudin-11 (dilution rate 1 : 1000; bs-21509R; Bioss), AMPK (Ser485) (1 : 1000 dilution; #2532; Cell Signaling Technology), SIRT3 (diluted at 1 : 1000; 10099-1-AP; Proteintech), β -actin (1 : 3,000 dilution; bs-0061R; Bioss), SOD2 (dilution rate 1 : 1000; bs-23402R; Bioss), NLRP3 (1 : 1000 dilution; 19771-1-AP; Proteintech), and IL-1 β (dilution rate 1 : 1000; #12703; Cell Signaling Technology) as the primary antibodies, as well as the HRP-labeled goat anti-rabbit antibody (diluted at 1 : 3000, bs-0295-HRP; Bioss) as the secondary antibody.

Techniques: In Vitro, Western Blot, Fluorescence, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Curcumin Ameliorates Age-Induced Tight Junction Impaired in Porcine Sertoli Cells by Inactivating the NLRP3 Inflammasome through the AMPK/SIRT3/SOD2/mtROS Signaling Pathway

doi: 10.1155/2023/1708251

Figure Lengend Snippet: Curcumin inhibits D-gal-induced decreased barrier function via inhibiting activated NLRP3 inflammasome and released IL-1 β in SCs in vitro . (a) Western blotting is carried out to analyze SOD2, NLRP3, and IL-1 β in porcine tissues. (b) SCs are treated for 48 h with D-gal (40 g/L), compound C (10 μ M), 3-TYP (50 μ M), and/or Curcumin (10 μ M). NLRP3 and IL-1 β proteins are explored through Western blotting. (c) SCs are treated with D-gal (40 g/L) and/or mito-TEMPO (50 μ M) for 48 h to analyze NLRP3 and IL-1 β proteins using Western blotting. (d) IL-1 β secretion is investigated by ELISA analysis using cell culture supernatant fractions. (e) SCs receive 48 h of treatment under D-gal (40 g/L), MCC950 (10 μ M), and/or IL-1Ra (20 ng/mL), followed by analysis of ZO-1, Occludin, and Claudin-11 proteins by virtue of Western blotting, with β -actin as the loading control. The mean ± SEM is used as the expression format for values; ∗ P < 0.05 and ∗∗ P < 0.01 vs. the control group; # P < 0.05 and ## P < 0.01 vs. the D-gal treatment group.

Article Snippet: After washing in ice-cold PBS, the acquired tissues and cells were processed with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer complemented with phenylmethylsulfonyl fluoride (PMSF, at 1 mM), followed by Western blotting in accordance with the previously mentioned methods [ ], with Occludin (diluted at 1 : 1000; bs-10011R; Bioss), ZO-1 (dilution rate 1 : 1000; bs-1329R; Bioss), p-AMPK (Ser485) (diluted at 1 : 1000; #2537; Cell Signaling Technology; Danvers; USA), Claudin-11 (dilution rate 1 : 1000; bs-21509R; Bioss), AMPK (Ser485) (1 : 1000 dilution; #2532; Cell Signaling Technology), SIRT3 (diluted at 1 : 1000; 10099-1-AP; Proteintech), β -actin (1 : 3,000 dilution; bs-0061R; Bioss), SOD2 (dilution rate 1 : 1000; bs-23402R; Bioss), NLRP3 (1 : 1000 dilution; 19771-1-AP; Proteintech), and IL-1 β (dilution rate 1 : 1000; #12703; Cell Signaling Technology) as the primary antibodies, as well as the HRP-labeled goat anti-rabbit antibody (diluted at 1 : 3000, bs-0295-HRP; Bioss) as the secondary antibody.

Techniques: In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Control, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Curcumin Ameliorates Age-Induced Tight Junction Impaired in Porcine Sertoli Cells by Inactivating the NLRP3 Inflammasome through the AMPK/SIRT3/SOD2/mtROS Signaling Pathway

doi: 10.1155/2023/1708251

Figure Lengend Snippet: Curcumin inhibits BTB disruption through inactivating the NLRP3 inflammasome via the SIRT3/AMPT/SOD2 signaling pathway in D-gal-treated murine testes. The mice were allocated into control group (group 1, no D-gal, without treatment), model group (group 2, with D-gal, without treatment), Curcumin-treated group (group 3, with 100 mg/kg Curcumin + D-gal), curcumin-treated group (group 4, with 200 mg/kg Curcumin + D-gal), and Curcumin-treated group (group 5, with 400 mg/kg Curcumin + D-gal). (a) Testicular weight change in the mice is detected. (b) The histological changes in testes were appraised with HE staining. The upper and lower panels manifest the changes in seminiferous tubules (original magnification 100×) and the magnified images of the boxed areas (original magnification 400×), respectively. (c) Typical immunofluorescence images for colocalizing ZO-1, Occludin, and Claudin-11 (green) by virtue of SOX9 (red) in the testes are displayed. (d) Western blotting is carried out to exploit ZO-1, Occludin, and Claudin-11 proteins. (e) p-AMPK, AMPK, SIRT3, SOD2, NLRP3, and IL-1 β proteins are examined using Western blotting. (f) Changes in SOD, MDA, and CAT content are examined. (g) The changes in sperm motility and deformity rate are measured. β -Actin is applied as the loading control, and the format of values is mean ± SEM; ∗ P < 0.05, ∗∗ P < 0.01 vs. the control group; # P < 0.05 and ## P < 0.01 vs. the D-gal treatment group.

Article Snippet: After washing in ice-cold PBS, the acquired tissues and cells were processed with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer complemented with phenylmethylsulfonyl fluoride (PMSF, at 1 mM), followed by Western blotting in accordance with the previously mentioned methods [ ], with Occludin (diluted at 1 : 1000; bs-10011R; Bioss), ZO-1 (dilution rate 1 : 1000; bs-1329R; Bioss), p-AMPK (Ser485) (diluted at 1 : 1000; #2537; Cell Signaling Technology; Danvers; USA), Claudin-11 (dilution rate 1 : 1000; bs-21509R; Bioss), AMPK (Ser485) (1 : 1000 dilution; #2532; Cell Signaling Technology), SIRT3 (diluted at 1 : 1000; 10099-1-AP; Proteintech), β -actin (1 : 3,000 dilution; bs-0061R; Bioss), SOD2 (dilution rate 1 : 1000; bs-23402R; Bioss), NLRP3 (1 : 1000 dilution; 19771-1-AP; Proteintech), and IL-1 β (dilution rate 1 : 1000; #12703; Cell Signaling Technology) as the primary antibodies, as well as the HRP-labeled goat anti-rabbit antibody (diluted at 1 : 3000, bs-0295-HRP; Bioss) as the secondary antibody.

Techniques: Disruption, Control, Staining, Immunofluorescence, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Protein Phosphatase 2A Improves Cardiac Functional Response to Ischemia and Sepsis

doi: 10.3390/ijms23094688

Figure Lengend Snippet: Differential mRNA expression. The basal cardiac gene expression profile of PP2A overexpressing (PP2A-TG) and littermate wild type (WT) mouse hearts was analyzed by a mouse genome gene chip. Data are presented as ratio of the mean PP2A-TG signal divided by the mean WT signal ± SD. More detailed data can be found in the and . ( A ) Several subunits of different protein phosphatases are summarized. ( B ) A selection of genes with various functions is shown. The abbreviations are: aldehyde dehydrogenase 2 (Aldh2), Ca 2+ calmodulin kinase II (CamKII), cardiac calsequestrin (CSQ2), endonuclease G (Endog), glycerin aldehyde phosphate dehydrogenase (GAPDH), heat shock protein 25 (HSP25), nitric oxide synthase 3 (NOS3), nucleoporin 62kDa (Nup62), proliferating cell nuclear antigen (PCNA), superoxide dismutase 2 (SOD2). Three RNA samples from each genotype (n = 3) were studied. * p < 0.05 vs. WT (by comparison of individual data sets).

Article Snippet: The following primary antibodies were used: calsequestrin (CSQ: rabbit polyclonal [#SP5340P], Acris Antibodies, Herford, Germany [now available from abcam #ab3516]); glyceraldehyde-3-phosphate dehydrogenase (GAPDH; mouse monoclonal [#ab9484], abcam, Cambridge, MA, USA); super oxide dismutase 2 (SOD2: rabbit polyclonal [#SPC-118C/D], StressMarq Biosciences, Victoria, Canada); Ca2+ calmodulin kinase II (CamKII: rabbit monoclonal [#2048-1], Epitomics, Burlingame, CA, U.S.A.); heat shock protein 25 (HSP25: rabbit polyclonal [#ADI-SPA-801], Enzo Life Science, Lörrach, Germany); heat shock protein 90 (HSP90: rat monoclonal [#ADI-SPA-845], Enzo Life Science, Lörrach, Germany); aldehyde dehydrogenase 2 (Aldh2; goat polyclonal [#ABIN571181], antibodies-online, Aachen, Germany); endonuclease G (Endog; goat polyclonal [sc-26923], Santa Cruz Biotechnology, Heidelberg, Germany); cathepsin B (rabbit polyclonal [#ABIN1002042], antibodies-online, Aachen, Germany); enolase3 (=enolase beta; rabbit polyclonal [ABIN310998], antibodies-online, Aachen, Germany); nucleoporin 62kDa (Nup62; rabbit polyclonal [#ABIN1013745], antibodies-online, Aachen, Germany); protein phosphatase 5 (PP5: mouse monoclonal [#611021], BD Transduction Laboratories, Heidelberg, Germany); regulatory A-subunit of protein phosphatase 2A (PP2A-A: goat polyclonal [#sc-6113], Santa Cruz Biotechnology, Heidelberg, Germany); catalytic subunit of protein phosphatase 2A (PP2A-C: rabbit monoclonal [#ab32141], abcam, Berlin, Germany); catalytic subunit alpha of protein phosphatase 1 (PP1c; mouse monoclonal [#sc-7482], Santa Cruz Biotechnology, Heidelberg, Germany); nitric oxide synthase 1 to 3 (NOS 1; NOS 2; NOS 3; rabbit polyclonal [#610310; #610333; #610299], BD Transduction Laboratories, Heidelberg, Germany).

Techniques: Expressing, Selection

Journal: International Journal of Molecular Sciences

Article Title: Protein Phosphatase 2A Improves Cardiac Functional Response to Ischemia and Sepsis

doi: 10.3390/ijms23094688

Figure Lengend Snippet: Basal cardiac protein expression. Basal cardiac protein expression of a selection of genes with various functions analyzed by Western blotting of wild type (WT) and PP2A overexpressing (PP2A-TG) mouse hearts. Original Western blots are shown in the . Quantification data of Western blots are presented as ratio of the mean PP2A-TG signal divided by the mean WT signal ± SD. The abbreviations are: aldehyde dehydrogenase 2 (Aldh2), cardiac calsequestrin (CSQ2), Ca 2+ calmodulin kinase II (CamKII), endonuclease G (Endog), glycerin aldehyde phosphate dehydrogenase (GAPDH), heat shock proteins 25 and 90 (HSP25, HSP90), nucleoporin 62kDa (Nup62), catalytic alpha subunit of protein phosphatase 1 (PP1c), structural A-subunit and catalytic C-subunit of protein phosphatase 2A (PP2A-A, PP2A-C), protein phosphatase 5 (PP5), superoxide dismutase 2 (SOD2). * p < 0.05 vs. WT.

Article Snippet: The following primary antibodies were used: calsequestrin (CSQ: rabbit polyclonal [#SP5340P], Acris Antibodies, Herford, Germany [now available from abcam #ab3516]); glyceraldehyde-3-phosphate dehydrogenase (GAPDH; mouse monoclonal [#ab9484], abcam, Cambridge, MA, USA); super oxide dismutase 2 (SOD2: rabbit polyclonal [#SPC-118C/D], StressMarq Biosciences, Victoria, Canada); Ca2+ calmodulin kinase II (CamKII: rabbit monoclonal [#2048-1], Epitomics, Burlingame, CA, U.S.A.); heat shock protein 25 (HSP25: rabbit polyclonal [#ADI-SPA-801], Enzo Life Science, Lörrach, Germany); heat shock protein 90 (HSP90: rat monoclonal [#ADI-SPA-845], Enzo Life Science, Lörrach, Germany); aldehyde dehydrogenase 2 (Aldh2; goat polyclonal [#ABIN571181], antibodies-online, Aachen, Germany); endonuclease G (Endog; goat polyclonal [sc-26923], Santa Cruz Biotechnology, Heidelberg, Germany); cathepsin B (rabbit polyclonal [#ABIN1002042], antibodies-online, Aachen, Germany); enolase3 (=enolase beta; rabbit polyclonal [ABIN310998], antibodies-online, Aachen, Germany); nucleoporin 62kDa (Nup62; rabbit polyclonal [#ABIN1013745], antibodies-online, Aachen, Germany); protein phosphatase 5 (PP5: mouse monoclonal [#611021], BD Transduction Laboratories, Heidelberg, Germany); regulatory A-subunit of protein phosphatase 2A (PP2A-A: goat polyclonal [#sc-6113], Santa Cruz Biotechnology, Heidelberg, Germany); catalytic subunit of protein phosphatase 2A (PP2A-C: rabbit monoclonal [#ab32141], abcam, Berlin, Germany); catalytic subunit alpha of protein phosphatase 1 (PP1c; mouse monoclonal [#sc-7482], Santa Cruz Biotechnology, Heidelberg, Germany); nitric oxide synthase 1 to 3 (NOS 1; NOS 2; NOS 3; rabbit polyclonal [#610310; #610333; #610299], BD Transduction Laboratories, Heidelberg, Germany).

Techniques: Expressing, Selection, Western Blot

Journal: Arthritis Research & Therapy

Article Title: Bach1 deficiency reduces severity of osteoarthritis through upregulation of heme oxygenase-1

doi: 10.1186/s13075-015-0792-1

Figure Lengend Snippet: Expression of microtubule-associated protein 1 light chain 3 ( LC3 ) and superoxide dismutase 2 ( SOD2 ) in articular cartilage. Knee joints from wild-type ( WT ) mice and Bach1 -/- mice were analyzed by immunohistochemistry for LC3 and SOD2. Total number of LC3- and SOD2-positive cells in six fields were counted and the percentage of positive cells was calculated. a Representative immunostaining of articular cartilage from 12-month- ( 12 M ) and 22 month ( 22 M )-old mice (aging model) (n = 4 per group). b Representative immunostaining of articular cartilage from a surgically induced osteoarthritis ( OA ) model (n = 4 per group). Original magnification × 20; scale bars 100 μm. Values are the mean ± SD. Statistical analysis was performed with the Mann–Whitney U test; * P <0.05, ** P <0.01 versus wild-type mice

Article Snippet: For anti-microtubule-associated protein 1 light chain 3 (LC3) antibody (1:100, AP1801a, ABGENT, San Diego, CA, USA), anti-manganese superoxide dismutase (MnSOD) antibody (1:100, SPC-117, StressMarq, Victoria, BC, Canada), sections in Immunoactive pH 6.0 (Matsunami Glass, Osaka, Japan) were heated in a microwave oven and kept at 85 °C for 1.5 minutes.

Techniques: Expressing, Immunohistochemistry, Immunostaining, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Bach1 deficiency reduces severity of osteoarthritis through upregulation of heme oxygenase-1

doi: 10.1186/s13075-015-0792-1

Figure Lengend Snippet: The involvement of heme oxygenase-1 ( HO-1 ) in changes observed in Bach1 -/- articular chondrocytes. Small interfering HO-1 ( siHO-1 ) or control siRNA (10 uM) was transfected into chondrocytes from wild-type ( WT ) and Bach1 -/- mice at 1 month of age (n = 4 per group). a The expression of superoxide dismutase 2 ( SOD2 ) protein was detected by immunoblot analysis. Values are the mean ± SD. Statistical analysis was performed with the Steel–Dwass test; * P <0.05 versus wild-type chondrocytes. b The expression of Mmp-13 and Adamts-5 in Bach1 -/- chondrocytes with IL-1β (1 ng/ml) for 24 h. Values are the mean ± SD. Statistical analysis was performed with the Steel test; * P <0.05 versus wild-type chondrocytes ( -IL ); # P <0.05 versus Bach1 -/- chondrocytes ( -IL ). c Caspase-3/7 was detected in wild-type and Bach1 -/- mouse chondrocytes with or without the oxidant tert-butyl hydroperoxide ( t-BHP ) (200 uM) for 5 h. Values are the mean ± SD. Statistical analysis was performed with the Steel–Dwass test; * P <0.05, ** P <0.01 versus wild-type chondrocytes with t-BHP; ## P <0.01 versus Bach1 -/- chondrocytes treated with t-BHP. GAPDH glyceraldehyde–3–phosphate dehydrogenase

Article Snippet: For anti-microtubule-associated protein 1 light chain 3 (LC3) antibody (1:100, AP1801a, ABGENT, San Diego, CA, USA), anti-manganese superoxide dismutase (MnSOD) antibody (1:100, SPC-117, StressMarq, Victoria, BC, Canada), sections in Immunoactive pH 6.0 (Matsunami Glass, Osaka, Japan) were heated in a microwave oven and kept at 85 °C for 1.5 minutes.

Techniques: Transfection, Expressing, Western Blot