Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Vascular EC-SOD limits the accumulation, proinflammatory profibrotic reprogramming, and hyaluronan binding of interstitial macrophages in hypoxia

doi: 10.1152/ajplung.00399.2024

Figure Lengend Snippet: Low vascular EC-SOD exacerbates hypoxia-induced interstitial macrophage accumulation. Flow cytometric analysis of lung interstitial macrophage (IM) numbers in mice expressing low vascular EC-SOD (EC-SOD SMC KO; EC-SOD loxp/loxp × Tg cre/SMMHC ) compared with floxed wild-type controls (WT; EC-SOD loxp/loxp ) at baseline (normoxia, Nx) and following 4 or 14 days of hypoxia exposure (Hx). Intravenous (IV) CD45 antibody administration was used to exclude intravascular cells as part of the “Dump” gate (gating shown in ). Total macrophages were determined as live IV-CD3 − B220 − Ly6G − CD45 + CD64 + singlets. IMs (CD64 + , CD11b hi CD11c low/int ) and resident alveolar macrophages (AM; CD64 + , CD11b hi CD11c low/int ) were separated based on CD11b and CD11c expression and IM1 (CD11c low MHCII low ), IM2 (CD11c low MHCII hi ), and IM3 (CD11c int MHCII hi ) subsets identified by the expression of CD11c and MHCII. Total macrophages ( A ), interaction (hypoxia:genotype) P = 0.01480; resident AMs ( B ), interaction (hypoxia:genotype) P = 0.02250; total IMs ( C ), interaction (hypoxia:genotype) P = 0.04467; IM1( D ), interaction (hypoxia:genotype) P = 0.02789; IM2 ( E ), interaction (hypoxia: genotype) P = 0.06856; and IM3 ( F ) counts, interaction (hypoxia:genotype) P = 0.04813 in whole lung over the hypoxia time course. Two-factor aligned rank test ANOVA, Tukey’s post hoc tests for comparisons, all P values where P < 0.2 are shown. n = 10–15 mice per group. Due to the SMMHC promotor’s location on the Y chromosome, all mice are male. All data are nonparametric and expressed as median ± interquartile range. EC-SOD, extracellular superoxide dismutase; KO, knockout; SMC, smooth muscle cell; SMMHC, SMC myosin heavy chain.

Article Snippet: Membranes were activated in methanol and blocked with 5% nonfat dry milk powder in Tris-buffered saline with 0.1% Tween 20 (TBST) for at least 1 h. After blocking, membranes were cut below the 75 kDa molecular weight marker and incubated overnight at 4°C with primary antibodies against EC-SOD (goat anti-mouse, R&D Systems, 1:800) or vinculin (rabbit anti-mouse, Cell Signaling Technology, 1:1,000) in 5% milkTBST.

Techniques: Expressing, Knock-Out

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Vascular EC-SOD limits the accumulation, proinflammatory profibrotic reprogramming, and hyaluronan binding of interstitial macrophages in hypoxia

doi: 10.1152/ajplung.00399.2024

Figure Lengend Snippet: Single dose of SOD mimetic before hypoxia attenuates pulmonary hypertension and vascular remodeling. A : chronic hypoxic pulmonary hypertension was assessed by direct right ventricle (RV) puncture and measurement of RV systolic pressure (RVSP). B : RV hypertrophy was assessed by right ventricle/left ventricle + septum weights. Muscularization of small pulmonary vessels after 21 days of hypoxia was evaluated using α-smooth muscle actin (α-SMA) antibody staining (purple) of lung sections from hypoxic mice treated with SOD mimetic or vehicle (PBS) compared with normoxic controls. Costaining was performed with HA-BP to also detect hyaluronan (brown). C : vessel muscularization at 21 days was quantified by counting the number of muscularized small vessels (<50 μm) with positive staining for α-SMA counted per high-power field (HPF). D : representative images taken at ×10 magnification (scale bars represent 200 μm) with muscularized vessels indicated with black arrows. One-way ANOVA, Tukey’s post hoc tests for multiple comparisons, n = 5–9 mice per group. Female mice indicated with closed symbols. All data expressed as means ± SD. HA-BP, hyaluronan-binding protein; Hx, hypoxia; Nx, normoxia; SOD, superoxide dismutase.

Article Snippet: Membranes were activated in methanol and blocked with 5% nonfat dry milk powder in Tris-buffered saline with 0.1% Tween 20 (TBST) for at least 1 h. After blocking, membranes were cut below the 75 kDa molecular weight marker and incubated overnight at 4°C with primary antibodies against EC-SOD (goat anti-mouse, R&D Systems, 1:800) or vinculin (rabbit anti-mouse, Cell Signaling Technology, 1:1,000) in 5% milkTBST.

Techniques: Staining, Binding Assay

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Vascular EC-SOD limits the accumulation, proinflammatory profibrotic reprogramming, and hyaluronan binding of interstitial macrophages in hypoxia

doi: 10.1152/ajplung.00399.2024

Figure Lengend Snippet: Perivascular Lyve1 + IMs are increased in EC-SOD SMC KO following 4 days of hypoxia and colocalize with HA. Lung sections were stained with a panel of antibodies to identify macrophage populations around pulmonary vessels in floxed WT controls and EC-SOD SMC KO mice at baseline (left hand side images) and during peak hypoxia-induced IM accumulation (4 D Hx; right hand side images). Immunofluorescence imaging was performed on the Vectra Polaris (Akoya) using inForm software. F480 was used to label all macrophages in white ( A ), CD163 in green ( B ), and Lyve1 in yellow ( C ) were used to label the Lyve1 + IM subset. α-SMA was used to identify smooth muscle (blue) in panels. Representative images showing increased interstitial macrophages in hypoxic WT and hypoxic EC-SOD SMC KO mice are shown from n = 3 mice per group; 40 magnification, scale bars 20 μm. White arrows indicate Lyve1-IMs and yellow arrows indicate Lyve1 + IMs in each section. D : adjacent lung sections were costained for α-SMA (purple) actin; EC-SOD, and HA-BP to detect perivascular hyaluronan (brown). Representative images taken at ×40 magnification (scale bars = 50 μm) from n = 3–5 mice per group. Due to the SMMHC promotor’s location on the Y chromosome, all mice used were male. α-SMA, α-smooth muscle actin; EC-SOD, extracellular superoxide dismutase; HA, hyaluronan; HA-BP, hyaluronan-binding protein; Hx, hypoxia; IMs, interstitial macrophages; KO, knockout; Lyve1, lymphatic vessel endothelial hyaluronan receptor 1; Nx, normoxia; SMC, smooth muscle cell; SMMHC, SMC myosin heavy chain; WT, wild type.

Article Snippet: Membranes were activated in methanol and blocked with 5% nonfat dry milk powder in Tris-buffered saline with 0.1% Tween 20 (TBST) for at least 1 h. After blocking, membranes were cut below the 75 kDa molecular weight marker and incubated overnight at 4°C with primary antibodies against EC-SOD (goat anti-mouse, R&D Systems, 1:800) or vinculin (rabbit anti-mouse, Cell Signaling Technology, 1:1,000) in 5% milkTBST.

Techniques: Staining, Immunofluorescence, Imaging, Software, Binding Assay, Knock-Out

Journal: Frontiers in Pharmacology

Article Title: Estrogen-Related Receptor γ Agonist DY131 Ameliorates Lipopolysaccharide-Induced Acute Liver Injury

doi: 10.3389/fphar.2021.626166

Figure Lengend Snippet: Primer sequence.

Article Snippet: Immunoblotting was performed using primary antibodies against ERRγ (Santa Cruz, sc-66883, 1:1,000), β-actin (Bioss, bs-0061R, 1:2,500), SOD1 (Proteintech, 67480-1-Ig, 1:5,000), SOD2 (Abclonal, A19576, 1:1,000), SOD3 (Abclonal, A6984, 1:1,000), cleaved caspase-3 (Cell Signaling Technology, 9,661, 1:1,000), and Bax (Cell Signaling Technology, 2,772, 1:1,000), followed by the incubation of peroxidase-conjugated goat anti-rabbit secondary antibody (Beyotime, A0208, 1:2,000).

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: Estrogen-Related Receptor γ Agonist DY131 Ameliorates Lipopolysaccharide-Induced Acute Liver Injury

doi: 10.3389/fphar.2021.626166

Figure Lengend Snippet: DY131 ameliorated oxidative stress in LPS-treated mice. (A,B) The levels of the antioxidant GSH and oxidative stress marker MDA in livers were measured using a commercial kit (n = 10 in each group). (C) mRNA expressions of SOD1, SOD2, and SOD3 were determined by qRT-PCR (n = 10 in each group). (D) Protein levels of SOD1, SOD2, and SOD3 were detected by Western blotting (n = 8 in each group). (E) Quantitative analyses of SOD1, SOD2, and SOD3 by densitometry (n = 8 in each group). (F) Representative images of DHE staining of liver tissues in different groups (magnification ×200, scale bar: 50 μm, n = 3 in each group). (G) Quantification of the mean fluorescence intensity of DHE was analyzed by ImageJ software. Data were presented as means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. the indicated group. NS, no significance.

Article Snippet: Immunoblotting was performed using primary antibodies against ERRγ (Santa Cruz, sc-66883, 1:1,000), β-actin (Bioss, bs-0061R, 1:2,500), SOD1 (Proteintech, 67480-1-Ig, 1:5,000), SOD2 (Abclonal, A19576, 1:1,000), SOD3 (Abclonal, A6984, 1:1,000), cleaved caspase-3 (Cell Signaling Technology, 9,661, 1:1,000), and Bax (Cell Signaling Technology, 2,772, 1:1,000), followed by the incubation of peroxidase-conjugated goat anti-rabbit secondary antibody (Beyotime, A0208, 1:2,000).

Techniques: Marker, Quantitative RT-PCR, Western Blot, Staining, Fluorescence, Software

Journal: Biology

Article Title: The H2S–Nrf2–Antioxidant Proteins Axis Protects Renal Tubular Epithelial Cells of the Native Hibernator Syrian Hamster from Reoxygenation-Induced Cell Death

doi: 10.3390/biology8040074

Figure Lengend Snippet: Reoxygenation increases the expression of SOD3, GR, ferritin H and xCT in the hamster, but not in mouse RPTECs. Hamster and mouse RPTECs were subjected to 24 h warm anoxia, washed with PBS, and then subjected to 2 h reoxygenation in fresh culture medium. Cell protein was extracted, and the expression of SOD3, GR, ferritin H, and xCT were assessed by western blotting. Panel A depicts three of the nine performed experiments. The changes in expression of the evaluated proteins followed the changes in Nrf2 levels. Compared to hamster RPTECs not subjected to anoxia and reoxygenation, the levels of SOD3, GR, ferritin H, and xCT after the 2 h of reoxygenation increased considerably. On the contrary, the expressions of SOD3, GR, ferritin H, and xCT did not alter significantly in mouse RPTECs ( B,C,D , E ). An asterisk corresponds to a p < 0.001 compared to the control cells, and error bars to SEM.

Article Snippet: Primary antibodies were rabbit polyclonal antibody against CBS (dilution: 1:1000, catalogue number TA338394, OriGene Technologies Inc., Rockville, MD, USA), mouse monoclonal against CSE (dilution: 1:100, catalogue number sc-374249, Santa Cruz Biotechnology, Dallas, TX, USA), mouse monoclonal antibody against 3-MST (dilution: 1:100, catalogue number sc-376168, Santa Cruz Biotechnology), rabbit polyclonal antibody against Nrf2 (dilution: 1:1000, catalogue number TA343586, OriGene Technologies), mouse monoclonal antibody against superoxide dismutase 3 (SOD3) (dilution: 1:100, catalogue number sc-271170, Santa Cruz Biotechnology), mouse monoclonal antibody against glutathione reductase (GR) (dilution: 1:100, catalogue number sc-133245, Santa Cruz Biotechnology), mouse monoclonal antibody against the ferritin heavy (H) chain (dilution: 1:100, catalogue number sc-376594, Santa Cruz Biotechnology), rabbit polyclonal antibody against cystine-glutamate antiporter (xCT) (dilution: 1:1000, catalogue number ANT-111, Alomone Labs, Jerusalem, Israel), and rabbit polyclonal antibody against β-actin (dilution: 1:2500, catalogue number 4967, Cell Signaling Technology, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Expressing, Western Blot, Control

Journal: Breast cancer research : BCR

Article Title: Vascular endothelial growth factor C promotes breast cancer progression via a novel antioxidant mechanism that involves regulation of superoxide dismutase 3.

doi: 10.1186/s13058-014-0462-2

Figure Lengend Snippet: Figure 5 Vascular endothelial growth factor C regulates Sod3 expression in 66 cl4 mammary carcinoma cells. (A) Total RNA from 66 cl4-scramble and 66 cl4-VEGF-C KD2 cells was converted to cDNA and used to perform a mouse oxidative stress PCR array. Nine candidate genes of the eighty-four examined were identified in the PCR array with more than a twofold change in response to VEGF-C KD. (B) Sod3 mRNA expression was determined using a real-time PCR SYBR Green assay on 66 cl4-scram and 66 cl4-VEGF-C KD1 and KD2 cells (top). Western blot analysis of Sod3 expression in 66 cl4-scram and VEGF-C KD1 and KD2 cells (bottom). (C) Sod3 mRNA expression was determined by real-time PCR SYBR Green assay on three pairs of 66 cl4-scram and VEGF-C KD1 or KD2 tumors (each pair was derived from the same animal).

Article Snippet: Antibody against human SOD3 was obtained from Novus Biologicals (NBP1-22417; Littleton, CO, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Western Blot, Derivative Assay

Journal: Breast cancer research : BCR

Article Title: Vascular endothelial growth factor C promotes breast cancer progression via a novel antioxidant mechanism that involves regulation of superoxide dismutase 3.

doi: 10.1186/s13058-014-0462-2

Figure Lengend Snippet: Figure 8 Expression of VEGFC and SOD3 in human cancers. (A) VEGFC and SOD3 expression values were retrieved from an Oncomine microarray data set [43] (as indicated in the figure) and were plotted by expression value. Statistical analysis was performed using Pearson r correlation (two-tailed). (B) Proposed model for the function of vascular endothelial growth factor C (VEGF-C) in breast cancer progression. Expression of VEGF-C in a subset of tumor cells confers the ability to resist oxidative stress generated during tumor growth, and this ability is partially mediated by Sod3. However, VEGF-C mediates other pathways that are important in conferring resistance to chemotherapies, thus contributing to tumor recurrence. Blocking VEGF-C signaling would therefore be expected to sensitize breast cancers to chemotherapies that induce oxidative stress, to contribute to a reduction in tumor-initiating cells (TICs) and to decrease lymphangiogenesis, thus likely improving survival and prevent recurrence.

Article Snippet: Antibody against human SOD3 was obtained from Novus Biologicals (NBP1-22417; Littleton, CO, USA).

Techniques: Expressing, Microarray, Two Tailed Test, Generated, Blocking Assay

Journal: European Journal of Histochemistry : EJH

Article Title: Analysis of Extracellular Superoxide Dismutase and Akt in Ascending Aortic Aneurysm With Tricuspid or Bicuspid Aortic Valve

doi: 10.4081/ejh.2014.2383

Figure Lengend Snippet: Immunohistochemical analysis of SOD3 protein. Immunostaining of SOD3 in nonaneurysmal ascending aortic media layer (A). Immunostaining of SOD3 in ascending aortic media layer from patients affected by AsAA associated to TAV (B) or BAV (C). Representative images are shown.

Article Snippet: Rabbit monoclonal antibody against GAPDH was purchased from Cell SignalTechnology Inc. (Danvers, MA, USA); mouse monoclonal antibody against human SOD3 was obtained from AbCam, (Cambridge, UK); rabbit polyclonal antibody against human p-Akt 1/2/3 (Ser473), rabbit polyclonal antibody against human Akt 1, goat polyclonal antibody against -actin , mouse monoclonal MMP-9 antibody, mouse monoclonal pErk antibody, rabbit polyclonal Erk antibody and each secondary antibody conjugated to horseradish peroxidase were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Immunohistochemical staining, Immunostaining

Journal: European Journal of Histochemistry : EJH

Article Title: Analysis of Extracellular Superoxide Dismutase and Akt in Ascending Aortic Aneurysm With Tricuspid or Bicuspid Aortic Valve

doi: 10.4081/ejh.2014.2383

Figure Lengend Snippet: Analysis of Erk1/Erk2 phosphorylation in the ascending aortic wall. Western blotting analysis, for SOD3 protein levels evaluation, was performed on ascending aorta protein extracts from six nonaneurysmal aortas (N), nine patients with AsAA associated to TAV and nine patients with AsAA associated to BAV (A); GAPDH , was used as loading control. Representative image is shown. Densitometric analysis of SOD3 protein levels (B). Data are reported as mean ± SD. *P<0.05.

Article Snippet: Rabbit monoclonal antibody against GAPDH was purchased from Cell SignalTechnology Inc. (Danvers, MA, USA); mouse monoclonal antibody against human SOD3 was obtained from AbCam, (Cambridge, UK); rabbit polyclonal antibody against human p-Akt 1/2/3 (Ser473), rabbit polyclonal antibody against human Akt 1, goat polyclonal antibody against -actin , mouse monoclonal MMP-9 antibody, mouse monoclonal pErk antibody, rabbit polyclonal Erk antibody and each secondary antibody conjugated to horseradish peroxidase were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Western Blot, Control