hyperoxia exposures  (Alion Pharmaceuticals)

Journal: Antioxidants

Article Title: Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury

doi: 10.3390/antiox11020212

Figure Lengend Snippet: Hyperoxia impairs alveolar macrophage-mediated efferocytosis, and Nrf2-deficiency worsens it in vivo. Nrf2 +/+ (WT) and Nrf2 −/− mice ( n = 3 per group) were exposed to room air or hyperoxia for 48 h, and a set of hyperoxia-exposed mice were allowed to recover at room air for 72 as outlined in schema. ( a ) BAL from these mice was obtained, and macrophages were incubated on cover glasses with apoptotic neutrophils for 1 h. Macrophages were washed to remove unbound/non-internalized apoptotic cells and stained with Diff Quick stain. Images were captured to quantify apoptotic neutrophil binding, and internalization (indicated by red arrows). ( b ) Representative images of macrophages with neutrophils for each experimental condition are shown. ( c ) The number of apoptotic neutrophils either bound or internalized by macrophages from ~5–9 fields were quantified and expressed as % efferocytosis. Versus room air of respective genotypes, † Nrf2 −/− versus WT counterparts, § hyperoxia vs. recovery; †† p < 0.01; ****/§§§§/†††† p < 0.0001. Purple, room air group; Red, hyperoxia group; Green, hyperoxia and recovery.

Article Snippet: Mice cages were placed in a hyperoxia exposure chamber (Cat # A30274, BioSpherix, Ltd., Parish, NY, USA).

Techniques: In Vivo, Incubation, Staining, Diff-Quik, Binding Assay

Journal: Antioxidants

Article Title: Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury

doi: 10.3390/antiox11020212

Figure Lengend Snippet: Nrf2 is required for optimal macrophage-mediated efferocytosis. BMDMФs from Nrf2 +/+ (WT) and Nrf2 −/− mice were cultured and exposed to hyperoxia for 24 h. ( a ) Macrophages (green) were then incubated with labeled apoptotic neutrophils (red) for 1 h, washed, and captured images. ( b ) Macrophages were incubated with green fluorescent beads. Representative images of macrophages with neutrophils or beads for each genotype and experimental condition are shown. Values represent from at least three independent samples. * Room air versus hyperoxia of respective genotypes, †, Nrf2 −/− versus WT counterparts. * p < 0.05; **/†† p < 0.01; **** p < 0.0001. Blue, room air group; Red, hyperoxia group.

Article Snippet: Mice cages were placed in a hyperoxia exposure chamber (Cat # A30274, BioSpherix, Ltd., Parish, NY, USA).

Techniques: Cell Culture, Incubation, Labeling

Journal: Antioxidants

Article Title: Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury

doi: 10.3390/antiox11020212

Figure Lengend Snippet: Nrf2-deficiency in neutrophils does not affect their engulfment by macrophages. Neutrophils from the bone marrow of wild-type (WT) and Nrf2 −/− mice were isolated, labelled, and subjected to apoptosis as detailed in methods. Apoptotic neutrophils (red) were incubated with WT BMDMФs and Nrf2 −/− BMDMФs for 1 h, and efferocytosis was quantified. ( a ) Representative images of macrophages with apoptotic neutrophils (red). The blue color represents DAPI. ( b ) quantification of efferocytosis. Values are from 3–4 fields of two independent samples. † p < 0.05; Nrf2 −/− versus WT counterparts. Blue, room air group; pink hyperoxia group.

Article Snippet: Mice cages were placed in a hyperoxia exposure chamber (Cat # A30274, BioSpherix, Ltd., Parish, NY, USA).

Techniques: Isolation, Incubation

Journal: Antioxidants

Article Title: Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury

doi: 10.3390/antiox11020212

Figure Lengend Snippet: Nrf2 regulates apoptotic neutrophil binding to the macrophages. Nrf2 +/+ (WT) and Nrf2 −/− BMDMФs were exposed to room air or hyperoxia and then incubated with cytochalasin D (CtyD) before adding the labeled apoptotic neutrophils. Efferocytosis was quantified. ( a ) Both bound/attached and internalized apoptotic cells in the presence and absence of CytD were enumerated, and data are represented % efferocytosis. * hyperoxia versus room air of respective genotypes, †, Nrf2 −/− versus WT counterparts; § DMSO versus CtyD of respective genotypes. */† p < 0.05; **/§§ p < 0.01; ***/†††/§§§ p < 0.001; §§§§ p < 0.0001. Blue, room air group; Red, hyperoxia group. ( b ) Both bound and internalized apoptotic cells were enumerated by comparing values of respective CytD (bound) and DMSO-treated (bound and internalized) samples, * Hyperoxia versus room air of respective genotypes, and §, Nrf2 −/− versus WT versus counterparts. Values are from three independent samples. * p < 0.05; **/§§ p < 0.01; §§§ p < 0.001. Blue, room air group; Red, hyperoxia group.

Article Snippet: Mice cages were placed in a hyperoxia exposure chamber (Cat # A30274, BioSpherix, Ltd., Parish, NY, USA).

Techniques: Binding Assay, Incubation, Labeling

Journal: Antioxidants

Article Title: Nrf2 Is Required for Optimal Alveolar-Macrophage-Mediated Apoptotic Neutrophil Clearance after Oxidant Injury

doi: 10.3390/antiox11020212

Figure Lengend Snippet: Nrf2 activation augments efferocytosis in hyperoxia exposed BMDMΦs. Nrf2 +/+ and Nrf2 −/− BMDMΦs were pretreated with CDDO-Im (20 nM) for 6 h and exposed to hyperoxia for 24 h along with CDDO-Im. After hyperoxia, BMDMΦs were incubated with CellTracker Red stained apoptotic neutrophils for 1 hour. Images were captured, and % efferocytosis was enumerated. Values are at least from three independent samples. * Hyperoxia versus room air of respective genotypes, † versus WT counterparts, and § DMSO versus CDDO of respective genotypes. § p < 0.05; ****/††††/§§§§ p < 0.0001. Blue, room air group; Red, hyperoxia group.

Article Snippet: Mice cages were placed in a hyperoxia exposure chamber (Cat # A30274, BioSpherix, Ltd., Parish, NY, USA).

Techniques: Activation Assay, Incubation, Staining

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: (A) Bronchoalveolar lavage (BAL) fluid analysis determined numbers of neutrophils for inflammation and total protein concentration for vascular permeability (n = 3/group for air and 48 h O2, n = 6/group for 72h O2), and (B) activity of lactate dehydrogenase (LDH) for cytotoxicity (n = 3/group for air, n = 4–5/group for O2). (C) Hyperoxia susceptibility determined by body weight loss was indicated as percent body weight change at the end of 72 h hyperoxia or air exposure (day 18) compared to the onset of the diet (day 1, n = 3/group for air, n = 6/group for O2). Data presented as group mean±SE. Two-way ANOVA used for all statistical analyses. *, P < 0.05 vs. genotype- and diet-matched air controls. +, P < 0.05 vs. diet- and exposure-matched Nrf2+/+ mice. §, P < 0.05 vs genotype- and exposure-matched AIN group.

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques: Protein Concentration, Permeability, Activity Assay

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: (A) Heat map from hierarchical clustering analysis depicts expression profiles of SFN-responded genes in Nrf2+/+ after 72 h hyperoxia (O2) exposure (n = 1,187, P < 0.01 with moderated t-test). Heat map for the same genes in Nrf2−/− mice were shown for comparison. Color bar indicates average expression intensity (n = 3/group) normalized to Nrf2+/+-PBS-Air group. (B) Top canonical pathways of lung genes significantly altered by SFN in air-exposed Nrf2+/+ (black bars) and in air-exposed Nrf2−/− mice (grey bars). (C) Mitochondrial oxidative phosphorylation complex is illustrated with genes (in red) that were induced by SFN treatment in Nrf2+/+ mice exposed to normoxia (room air). (D) Top diseases and bio-functions of SFN-responsive lung genes in Nrf2+/+ mice included energy metabolism such as fatty acid beta-oxidation. (E) Lung genes significantly reduced by SFN in Nrf2+/+ mice were involved in the network of organismal injury and abnormality (scores 32–41), in which key molecules such as TNF receptor associated factor 1 (Traf1) and multiple mitogen-activated protein kinase (MAPK) cascade enzymes (e.g., Map3k8) were predicted to play central roles with NF-κB. Analysis was done by Ingenuity Pathway Analysis software.

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques: Expressing, Comparison, Phospho-proteomics, Software

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: (A) Pathway analysis for hyperoxia-responsive genes in PBS-received Nrf2+/+ mice (n= 7162 genes, P < 0.01, Moderated t-test) demonstrated p53 as a key upstream regulator for the hyperoxia-altered lung genes, which may sequentially modulate other signal transducers. (B) In Nrf2−/− mice that received PBS, O2 altered genes (n = 4,799, P < 0.01) involved predominantly in IL-17A signaling pathway, which may lead to severe neutrophil infiltration. (C) Nrf2-dependently modulated genes during hyperoxia (n = 816, P < 0.01) such as Selp and Fcgr2b may contribute to the differential lung edema between Nrf2+/+ and Nrf2−/− mice given PBS.

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques:

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: (A) Top canonical pathways of Nrf2-dependently changed gene transcripts with PBS (top, gray bars) or SFN (bottom, black bars) pretreatment in response to hyperoxia. (B) Profile analysis classified Nrf2-dependently regulated genes by similar expression patterns. (C) Pathway analysis for SFN-responsive genes in hyperoxia-exposed Nrf2−/− mice (n= 533, P < 0.01) depicted that genes altered by SFN only in these mice (e.g., Sele, Itga5, Lif, Flnb) may stimulate cellular movement and interaction by activating cell spreading, attachment, and homing, through which SFN may exert Nrf2-independent responses against hyperoxia in Nrf2−/− mice. Analyses were done using Ingenuity Pathway Analysis and GeneSpring software.

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques: Expressing, Software

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: Mitochondrial genome copy numbers determined by droplet digital PCR (ddPCR).

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques: Digital PCR

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: (A) Immunohistochemical localization of voltage-dependent anion-selective channel 1 (VDAC1)/porin, a mitochondrial membrane potential marker, in lung tissue sections. Brown dots indicate VDAC1-positive cells. Representative light photomicrographs are shown (n = 3–4/group). VDAC1 localization at baseline lung (PBS/Air) is indicated by small arrows. Thick black arrows indicate areas with increased VDAC1 expression compared to genotype-matched PBS/Air. White arrows indicate areas with decreased VDAC1 compared to genotype-matched PBS/Air. SFN = sulforaphane. O2 = Hyperoxia. AV, alveoli; BR, bronchi; BV, blood vessel; PA, pulmonary artery; TB, terminal bronchiole. Bar = 100 μm. (B) Aliquots of lung cytosolic proteins were subjected for Western blotting using specific antibodies. Representative images from multiple analyses of pooled proteins (n = 3/antibody) presented. ATP5A = ATP synthase subunit alpha, mitochondrial. MT-CO1 = mitochondrially encoded cytochrome c oxidase subunit 1. SDH8 = succinate dehydrogenase subunit B. NDUFB8 = NADH dehydrogenase (Ubiquinone) 1 beta subcomplex, 8. VDAC1 = Voltage-dependent anion-selective channel 1. PPLA=Cardiac phospholamban. kDa = kilodalton. Scanned band images were quantitated by densitometry. Data presented as group mean ± SE. Two-way ANOVA used for all statistical analyses. *, P < 0.05 vs. genotype- and pretreatment-matched air controls. +, P < 0.05 vs. pretreatment- and exposure-matched Nrf2+/+ mice. §, P < 0.05 vs. genotype- and exposure-matched PBS group. (C) Aliquots of pooled lung nuclear protein (5 μg) were incubated with an end-labeled oligonucleotide probe containing antioxidant response element (ARE) consensus sequence, and gel shift analysis determined total ARE binding. Nuclear proteins (5 μg) from PBS/hyperoxia-Nrf2−/− mice were run as a negative control. Nuclear proteins were subjected for Western blot analysis using Nrf2-specific antibody and images were quantified. *, P < 0.05 vs. pretreatment-matched air controls. §, P < 0.05 vs. PBS/hyperoxia group. (D) Aliquots of pooled lung nuclear protein (5 μg) were incubated with an end-labeled oligonucleotide probe containing NF-κB consensus sequence, and gel shift analysis determined total NF-κB binding. Two shifted bands (arrow heads) indicate total DNA-NF-κB complex. Specific activity for p65 NF-κB subunit was quantified using a transcription factor ELISA. Nuclear proteins from PBS/hyperoxia-Nrf2−/− mice were used for reaction with cold probes (20 pmol addition of oligonucleotide) and for no antibody control to verify the reaction specificity. *, P < 0.05 vs. genotype- and pretreatment-matched air controls. +, P < 0.05 vs. pretreament- and exposure-matched Nrf2+/+ mice. §, P < 0.05 vs. genotype- and exposure-matched PBS group.

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques: Immunohistochemical staining, Membrane, Marker, Expressing, Western Blot, Incubation, Labeling, Sequencing, Gel Shift, Binding Assay, Negative Control, Activity Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium *

doi: 10.1074/jbc.M111.294546

Figure Lengend Snippet: Role of nmMLCK in hyperoxia-mediated ROS and superoxide production in HPAECs. HPAECs grown on 35-mm dishes (∼90% confluence) were pretreated with increasing concentrations of ML-7 for 30 min and exposed to normoxia or hyperoxia for 3 h, and total ROS and superoxide accumulations were measured by DCFDA and hydroethidine immunofluorescence (A and B). In C, E, F, and G, HPAECs were transfected with scrambled (scRNA) or nmMLCK siRNA (50 nm) for 72 h as described under “Experimental Procedures.” Values for ROS and superoxide production are the mean ± S.D. from three independent experiments done in triplicate. *, significantly different from normoxia (p < 0.05); **, significantly different from untreated hyperoxia (p < 0.01). Total RNA was isolated from scrambled and nmMLCK siRNA-transfected cells, and real-time PCR was performed in a Light Cycler using SYBR Green QuantiTect (C). Data are from three independent experiments and are expressed as relative gene expression normalized to 18 S RNA. In D and E, cell lysates (20 μg of proteins) from ML-7, scrambled or nmMLCK siRNA-transfected cells were subjected to SDS-PAGE on 10% precast Tris-glycine gels and Western-blotted (IB) with anti-nmMLCK, phospho-MLC, and MLC antibodies. Shown is a representative blot of three independent experiments. In F and G, HPAECs were transfected with scrambled RNA or nmMLCK siRNA (50 nm) for 72 h, and cells were loaded with 10 μm DCFDA (F) or hydroethidine (G) for 30 min before exposure to normoxia or hyperoxia for 3 h. Formation of total ROS (F) and superoxide (G) was quantified as described above. Values are the mean ± S.D. of three independent experiments in triplicate. *, significantly different from normoxia (N) (p < 0.05); **, significantly different from hyperoxia exposure (HO) (p < 0.01).

Article Snippet: Antibodies for MLCK, MLC, cortactin, and p47, protein A/G plus agarose, and BSA were from Santa Cruz Biotechnology (Santa Cruz, CA); scrambled RNA and siRNA MLCK were from Dharmacon (Lafayette, CO); shMLCK-GFP was from Open Biosystems (Huntsville, AL); adenoviral construct nmMLCK-FLAG (WT) was generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA); Rac1 antibody was from BD Biosciences; Gene silencer was from Genlantis (San Diego, CA); FuGENE HD transfection reagent was from Roche Applied Science; phosphatase inhibitor mixture, ML-7, and antibodies for actin, phospho-Src (Tyr-418), and FLAG were from Sigma; anti-phospho-cortactin (Tyr-486) antibody was from Chemicon (Boronia, Australia); microscopy Lab-Tek slide chambers were from Electron Microscopy Sciences (Hatfield, PA); glass bottom 35-mm dishes were from MatTek Corp. (Ashland, MA); incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA); cell lysis buffer and antibodies for p-MLC and caveolin-1 were from Cell Signaling Technology (Danvers, MA).

Techniques: Immunofluorescence, Transfection, Isolation, Real-time Polymerase Chain Reaction, SYBR Green Assay, Expressing, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium *

doi: 10.1074/jbc.M111.294546

Figure Lengend Snippet: Overexpression of nmMLCK potentiates hyperoxia-induced ROS and superoxide production in HPAECs. HPAECs grown on 35-mm dishes (∼60% confluence) were transfected with vector control or FLAG-tagged nmMLCK WT (1 μg/ml) using FuGENE HD (3 μg/ml) transfection reagent for 72 h as described under “Experimental Procedures.” A, cell lysates (20 μg of proteins) from vector control and FLAG-tagged nmMLCK transfected cells were subjected to SDS-PAGE and immunoblotted with anti-MLCK, anti-FLAG and anti-actin antibodies. A representative immunoblot from three independent experiments is shown. B and C, vector control and FLAG-tagged nmMLCK (WT)-transfected cells were loaded with 10 μm DCFDA (B) or hydroethidine (C) for 30 min before exposure to normoxia (N) or HO for 3 h. Formation of total ROS (B) and superoxide (C) were quantified as described under “Experimental Procedures.” Values are the mean ± S.D. of three independent experiments in triplicate. *, significantly different from normoxia (N) (p < 0.05); **, significantly different from vector control cells exposed to hyperoxia (HO) (p < 0.001).

Article Snippet: Antibodies for MLCK, MLC, cortactin, and p47, protein A/G plus agarose, and BSA were from Santa Cruz Biotechnology (Santa Cruz, CA); scrambled RNA and siRNA MLCK were from Dharmacon (Lafayette, CO); shMLCK-GFP was from Open Biosystems (Huntsville, AL); adenoviral construct nmMLCK-FLAG (WT) was generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA); Rac1 antibody was from BD Biosciences; Gene silencer was from Genlantis (San Diego, CA); FuGENE HD transfection reagent was from Roche Applied Science; phosphatase inhibitor mixture, ML-7, and antibodies for actin, phospho-Src (Tyr-418), and FLAG were from Sigma; anti-phospho-cortactin (Tyr-486) antibody was from Chemicon (Boronia, Australia); microscopy Lab-Tek slide chambers were from Electron Microscopy Sciences (Hatfield, PA); glass bottom 35-mm dishes were from MatTek Corp. (Ashland, MA); incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA); cell lysis buffer and antibodies for p-MLC and caveolin-1 were from Cell Signaling Technology (Danvers, MA).

Techniques: Over Expression, Transfection, Plasmid Preparation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium *

doi: 10.1074/jbc.M111.294546

Figure Lengend Snippet: nmMLCK siRNA attenuates hyperoxia-induced phosphorylation of MLC and cortactin and actin rearrangement in HPAECs. HPAECs grown on 8-well slide chambers were transfected with scrambled RNA or nmMLCK siRNA (50 nm) for 72 h before exposure to either normoxia (N) or HO for 3 h, washed, fixed, permeabilized, and probed with anti-phospho-cortactin, anti-phospho-MLC antibodies, or phalloidin for actin staining. nmMLCK siRNA blunted hyperoxia-induced phosphorylation of cortactin and MLC near the cell periphery and enhanced actin stress fibers under both normoxia and hyperoxia as determined by immunofluorescent microscopy. A representative immunofluorescence image from three independent experiments is shown.

Article Snippet: Antibodies for MLCK, MLC, cortactin, and p47, protein A/G plus agarose, and BSA were from Santa Cruz Biotechnology (Santa Cruz, CA); scrambled RNA and siRNA MLCK were from Dharmacon (Lafayette, CO); shMLCK-GFP was from Open Biosystems (Huntsville, AL); adenoviral construct nmMLCK-FLAG (WT) was generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA); Rac1 antibody was from BD Biosciences; Gene silencer was from Genlantis (San Diego, CA); FuGENE HD transfection reagent was from Roche Applied Science; phosphatase inhibitor mixture, ML-7, and antibodies for actin, phospho-Src (Tyr-418), and FLAG were from Sigma; anti-phospho-cortactin (Tyr-486) antibody was from Chemicon (Boronia, Australia); microscopy Lab-Tek slide chambers were from Electron Microscopy Sciences (Hatfield, PA); glass bottom 35-mm dishes were from MatTek Corp. (Ashland, MA); incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA); cell lysis buffer and antibodies for p-MLC and caveolin-1 were from Cell Signaling Technology (Danvers, MA).

Techniques: Transfection, Staining, Microscopy, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium *

doi: 10.1074/jbc.M111.294546

Figure Lengend Snippet: Down-regulation of nmMLCK with nmMLCK siRNA blunted hyperoxia-induced translocation and co-localization of p47phox with cortactin. HPAECs grown on 8-well slide chambers were transfected with scrambled siRNA or MLCK siRNA for 72 h then exposed to normoxia or hyperoxia (3 h), washed, fixed, permeabilized, probed with anti-cortactin or anti-p47phox antibodies, and examined by immunofluorescence microscopy using a 60× oil objective. The cortactin (red) and p47phox (green) images show matched cell fields for each condition. Exposure of cells to hyperoxia resulted in redistribution of cortactin and p47phox to the cell periphery, whereas MLCK siRNA blunted cortactin and p47phox redistribution and co-localization (yellow in merged images). A representative image from three independent experiments is shown.

Article Snippet: Antibodies for MLCK, MLC, cortactin, and p47, protein A/G plus agarose, and BSA were from Santa Cruz Biotechnology (Santa Cruz, CA); scrambled RNA and siRNA MLCK were from Dharmacon (Lafayette, CO); shMLCK-GFP was from Open Biosystems (Huntsville, AL); adenoviral construct nmMLCK-FLAG (WT) was generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA); Rac1 antibody was from BD Biosciences; Gene silencer was from Genlantis (San Diego, CA); FuGENE HD transfection reagent was from Roche Applied Science; phosphatase inhibitor mixture, ML-7, and antibodies for actin, phospho-Src (Tyr-418), and FLAG were from Sigma; anti-phospho-cortactin (Tyr-486) antibody was from Chemicon (Boronia, Australia); microscopy Lab-Tek slide chambers were from Electron Microscopy Sciences (Hatfield, PA); glass bottom 35-mm dishes were from MatTek Corp. (Ashland, MA); incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA); cell lysis buffer and antibodies for p-MLC and caveolin-1 were from Cell Signaling Technology (Danvers, MA).

Techniques: Translocation Assay, Transfection, Immunofluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium *

doi: 10.1074/jbc.M111.294546

Figure Lengend Snippet: Overexpression of MLCK-FLAG wild type enhances hyperoxia-induced association of nmMLCK with cortactin and p47phox in cell periphery. HPAECs grown on 8-well slide chambers were transfected with vector control or FLAG-tagged MLCK wild type (1 μg/ml) and FuGENE HD (3 μg/ml) transfection reagent for 72 h and then exposed to either normoxia (N) or HO for 3 h. Cells were, washed, fixed, permeabilized, and probed with anti-cortactin (A), anti-p47phox (B), or anti-FLAG (A and B) antibodies and examined by immunofluorescence microscopy using a 60× oil objective. Exposure of cells to hyperoxia resulted in redistribution of cortactin and p47phox to the cell periphery with enhanced nmMLCK association and co-localization (yellow in merged images). Shown is a representative image from three independent experiments. C and D represent semiquantitation of the co-localization using an image analyzer showed an ∼2.0-fold increase in co-localization (yellow) between nmMLCK (red) and cortactin (green) and an ∼2.5-fold increase in co-localization between nmMLCK and p47phox.

Article Snippet: Antibodies for MLCK, MLC, cortactin, and p47, protein A/G plus agarose, and BSA were from Santa Cruz Biotechnology (Santa Cruz, CA); scrambled RNA and siRNA MLCK were from Dharmacon (Lafayette, CO); shMLCK-GFP was from Open Biosystems (Huntsville, AL); adenoviral construct nmMLCK-FLAG (WT) was generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA); Rac1 antibody was from BD Biosciences; Gene silencer was from Genlantis (San Diego, CA); FuGENE HD transfection reagent was from Roche Applied Science; phosphatase inhibitor mixture, ML-7, and antibodies for actin, phospho-Src (Tyr-418), and FLAG were from Sigma; anti-phospho-cortactin (Tyr-486) antibody was from Chemicon (Boronia, Australia); microscopy Lab-Tek slide chambers were from Electron Microscopy Sciences (Hatfield, PA); glass bottom 35-mm dishes were from MatTek Corp. (Ashland, MA); incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA); cell lysis buffer and antibodies for p-MLC and caveolin-1 were from Cell Signaling Technology (Danvers, MA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium *

doi: 10.1074/jbc.M111.294546

Figure Lengend Snippet: Down-regulation of nmMLCK with nmMLCK siRNA blunted the hyperoxia-induced association of cortactin with Src and p47phox and phosphorylation of cortactin, Src, and p47phox in HPAECs. A, HPAECs grown in 100-mm dishes to ∼90% confluence were exposed to either normoxia (N) or HO for 3 h. Cell lysates (500 μg proteins) from normoxia- or HO-exposed cells were immunoprecipitated (IP) with anti-nmMLCK antibody as described under “Experimental Procedures.” Immunoprecipitates were analyzed by 10% SDS-PAGE and probed with anti-nmMLCK, anti-cortactin, anti-Src, anti-p47phox, anti-actin, and anti-MLC antibodies. A representative blot from three independent experiments is shown. IB, immunoblot. B, HPAECs grown in 100-mm dishes to ∼50% confluence were transfected with scrambled (sc) RNA or nmMLCK siRNA (50 nm) for 72 h, and down-regulation of nmMLCK expression was verified by Western blotting as described under “Experimental Procedures.” Total cell lysates (500 μg of proteins) from scrambled and nmMLCK siRNA-treated cells were subjected to immunoprecipitation with anti-cortactin antibody, and immunoprecipitates were analyzed by 10% SDS-PAGE and probed with anti-cortactin (equal loading), anti-Src, anti-p47phox, anti-phosphotyrosine and anti-MLC antibodies. A representative blot from three independent experiments is shown.

Article Snippet: Antibodies for MLCK, MLC, cortactin, and p47, protein A/G plus agarose, and BSA were from Santa Cruz Biotechnology (Santa Cruz, CA); scrambled RNA and siRNA MLCK were from Dharmacon (Lafayette, CO); shMLCK-GFP was from Open Biosystems (Huntsville, AL); adenoviral construct nmMLCK-FLAG (WT) was generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA); Rac1 antibody was from BD Biosciences; Gene silencer was from Genlantis (San Diego, CA); FuGENE HD transfection reagent was from Roche Applied Science; phosphatase inhibitor mixture, ML-7, and antibodies for actin, phospho-Src (Tyr-418), and FLAG were from Sigma; anti-phospho-cortactin (Tyr-486) antibody was from Chemicon (Boronia, Australia); microscopy Lab-Tek slide chambers were from Electron Microscopy Sciences (Hatfield, PA); glass bottom 35-mm dishes were from MatTek Corp. (Ashland, MA); incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA); cell lysis buffer and antibodies for p-MLC and caveolin-1 were from Cell Signaling Technology (Danvers, MA).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium *

doi: 10.1074/jbc.M111.294546

Figure Lengend Snippet: nmMLCK regulates hyperoxia-induced enrichment of cortactin, Src, and p47phox in caveolin-enriched microdomains. HPAECs grown in 100-mm dishes were transfected with scrambled (sc) RNA or nmMLCK siRNA (50 nm) for 72 h (A) or pretreated with ML-7 (1 μm, 30 min) (B) before exposure to either normoxia (N) or HO for 3 h, and caveolin-enriched microdomains were isolated as described under “Experimental Procedures.” Samples were then analyzed by 4–20% SDS-PAGE and probed with anti-cortactin, anti-Src, anti-p47phox, anti-MLC, anti-actin, anti-phospho-cortactin, anti-phospho-Src, and anti-phospho-MLC antibodies. Immunoblots (IB) were quantified by ImageJ software and expressed as a ratio to total caveolin-1. Values are the mean ± S.D. from three independent experiments. *, significantly different from normoxia (p < 0.05); **, significantly different from scrambled siRNA transfected cells exposed to hyperoxia.

Article Snippet: Antibodies for MLCK, MLC, cortactin, and p47, protein A/G plus agarose, and BSA were from Santa Cruz Biotechnology (Santa Cruz, CA); scrambled RNA and siRNA MLCK were from Dharmacon (Lafayette, CO); shMLCK-GFP was from Open Biosystems (Huntsville, AL); adenoviral construct nmMLCK-FLAG (WT) was generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA); Rac1 antibody was from BD Biosciences; Gene silencer was from Genlantis (San Diego, CA); FuGENE HD transfection reagent was from Roche Applied Science; phosphatase inhibitor mixture, ML-7, and antibodies for actin, phospho-Src (Tyr-418), and FLAG were from Sigma; anti-phospho-cortactin (Tyr-486) antibody was from Chemicon (Boronia, Australia); microscopy Lab-Tek slide chambers were from Electron Microscopy Sciences (Hatfield, PA); glass bottom 35-mm dishes were from MatTek Corp. (Ashland, MA); incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA); cell lysis buffer and antibodies for p-MLC and caveolin-1 were from Cell Signaling Technology (Danvers, MA).

Techniques: Transfection, Isolation, SDS Page, Western Blot, Software

Journal: The Journal of Biological Chemistry

Article Title: Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium *

doi: 10.1074/jbc.M111.294546

Figure Lengend Snippet: nmMLCK-KO mice exhibit reduced lung injury, leakage, and inflammation and ROS production in vivo. Male C57BL/6 WT or nmMLCK knock out (−/−) mice in the same background were exposed to either normoxia (N) or HO for 72 h. At the end of the experiment mice were anesthetized, BAL fluid was collected, lungs were perfused with fresh PBS several times, and whole lungs without trachea were paraffin-embedded. A–C, paraffin-embedded lung tissues from normoxia or HO animals were, sectioned, and immunostained with anti-phospho-MLC (A), anti-phospho-Src (B), or anti-phospho-cortactin (C) antibodies and examined under immunofluorescence microscopy using 60× oil objective. D, lung tissues from normoxia or HO-exposed animals were stored in formalin for 24 h before processing for staining with hematoxylin and eosin and lung morphology was evaluated using 40 X objective. E, BAL fluid collected from normoxia- or HO-exposed animals were subjected to cytospin, and differential cell counts were performed. Shown is a graphic representation of macrophages (M) and neutrophils (N) in BAL fluid from wild type and nmMLCK−/− mice exposed to normoxia or hyperoxia. Values are the mean ± S.D. from three independent experiments. *, significantly different from animals exposed to normoxia (p < 0.05); **, significantly different from animals exposed to normoxia (p < 0.01); ***, significantly different from wild type mice exposed to hyperoxia (p < 0.005). F and G, BAL fluid from mice exposed to normoxia or hyperoxia was analyzed for H2O2 (F) and total protein (G). Values are the mean ± S.D. from three independent experiments. *, significantly different from animals exposed to normoxia (p < 0.05); **, significantly different from wild type animals exposed to hyperoxia (p < 0.01).

Article Snippet: Antibodies for MLCK, MLC, cortactin, and p47, protein A/G plus agarose, and BSA were from Santa Cruz Biotechnology (Santa Cruz, CA); scrambled RNA and siRNA MLCK were from Dharmacon (Lafayette, CO); shMLCK-GFP was from Open Biosystems (Huntsville, AL); adenoviral construct nmMLCK-FLAG (WT) was generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA); Rac1 antibody was from BD Biosciences; Gene silencer was from Genlantis (San Diego, CA); FuGENE HD transfection reagent was from Roche Applied Science; phosphatase inhibitor mixture, ML-7, and antibodies for actin, phospho-Src (Tyr-418), and FLAG were from Sigma; anti-phospho-cortactin (Tyr-486) antibody was from Chemicon (Boronia, Australia); microscopy Lab-Tek slide chambers were from Electron Microscopy Sciences (Hatfield, PA); glass bottom 35-mm dishes were from MatTek Corp. (Ashland, MA); incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA); cell lysis buffer and antibodies for p-MLC and caveolin-1 were from Cell Signaling Technology (Danvers, MA).

Techniques: In Vivo, Knock-Out, Immunofluorescence, Microscopy, Staining

Journal: The Journal of Biological Chemistry

Article Title: Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium *

doi: 10.1074/jbc.M111.294546

Figure Lengend Snippet: Overexpression of FLAG-tagged nmMLCK wild type in nmMLCK-deficient mouse lung ECs restores hyperoxia-induced ROS/O2˙̄ production. Mouse lung endothelial cells isolated from 4-5-week-old C57BL/6 and nmMLCK−/− null mice were isolated using collagenase A as described under “Experimental Procedures.” In A and B, lungs ECs grown to ∼80% confluence from wild type and nmMLCK−/− mice were exposed to normoxia or hyperoxia for 3 h, and total ROS and superoxide generated in cells were quantified as described under “Experimental Procedures.” Values for ROS and superoxide production are the mean ± S.D. from three independent experiments done in triplicate. *, significantly different from normoxia (p < 0.01); **, significantly different from untreated hyperoxia (p < 0.05). In C and D, lung ECs from nmMLCK−/− null mice were infected with vector control or adenoviral human nmMLCK (AdMLCK (WT)) (5 pfu/cell) in EBM-2 complete medium for 24 h before exposure to normoxia or hyperoxia for 3 h. Total ROS and superoxide accumulation in cells were measured by DCFDA and hydroethidine (HE) fluorescence. Values for ROS and superoxide production are the mean ± S.D. from three independent experiments done in triplicate. *, significantly different adenoviral nmMLCK wild type-infected cells exposed to normoxia (p < 0.01). In E, cell lysates (20 μg proteins) from wild type, nmMLCK−/−, and FLAG-tagged human nmMLCK-infected mouse lung ECs were subjected to SDS-PAGE and immunoblotted with anti-MLCK, anti-FLAG, and anti-actin antibodies. A representative immunoblot (IB) from three independent experiments is shown. Immunoblots were analyzed by densitometry and quantified.

Article Snippet: Antibodies for MLCK, MLC, cortactin, and p47, protein A/G plus agarose, and BSA were from Santa Cruz Biotechnology (Santa Cruz, CA); scrambled RNA and siRNA MLCK were from Dharmacon (Lafayette, CO); shMLCK-GFP was from Open Biosystems (Huntsville, AL); adenoviral construct nmMLCK-FLAG (WT) was generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA); Rac1 antibody was from BD Biosciences; Gene silencer was from Genlantis (San Diego, CA); FuGENE HD transfection reagent was from Roche Applied Science; phosphatase inhibitor mixture, ML-7, and antibodies for actin, phospho-Src (Tyr-418), and FLAG were from Sigma; anti-phospho-cortactin (Tyr-486) antibody was from Chemicon (Boronia, Australia); microscopy Lab-Tek slide chambers were from Electron Microscopy Sciences (Hatfield, PA); glass bottom 35-mm dishes were from MatTek Corp. (Ashland, MA); incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA); cell lysis buffer and antibodies for p-MLC and caveolin-1 were from Cell Signaling Technology (Danvers, MA).

Techniques: Over Expression, Isolation, Generated, Infection, Plasmid Preparation, Fluorescence, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium *

doi: 10.1074/jbc.M111.294546

Figure Lengend Snippet: Overexpression of FLAG-tagged nmMLCK wild type in nmMLCK-deficient mouse lung ECs restores hyperoxia-induced redistribution of p-MLC, p-Src, and p-cortactin to cell periphery. Mouse lung ECs isolated from 4–5-week-old C57BL/6 wild type and nmMLCK−/− mice were grown on 8-well slide chambers to ∼80% confluence. In some experiments ECs isolated from nmMLCK−/− null mice were infected with vector control or adenoviral human nmMLCK wild type (AdMLCK, 5 pfu/cell) for 24 h. Cells were exposed to normoxia (N) or HO for 3 h, washed, fixed, permeabilized, and probed with anti-phospho-MLC (A), anti-phospho-Src (B), or anti-phospho-cortactin (C) antibodies and examined by immunofluorescence microscopy using a 60× oil objective. A representative image from three independent experiments is shown.

Article Snippet: Antibodies for MLCK, MLC, cortactin, and p47, protein A/G plus agarose, and BSA were from Santa Cruz Biotechnology (Santa Cruz, CA); scrambled RNA and siRNA MLCK were from Dharmacon (Lafayette, CO); shMLCK-GFP was from Open Biosystems (Huntsville, AL); adenoviral construct nmMLCK-FLAG (WT) was generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA); Rac1 antibody was from BD Biosciences; Gene silencer was from Genlantis (San Diego, CA); FuGENE HD transfection reagent was from Roche Applied Science; phosphatase inhibitor mixture, ML-7, and antibodies for actin, phospho-Src (Tyr-418), and FLAG were from Sigma; anti-phospho-cortactin (Tyr-486) antibody was from Chemicon (Boronia, Australia); microscopy Lab-Tek slide chambers were from Electron Microscopy Sciences (Hatfield, PA); glass bottom 35-mm dishes were from MatTek Corp. (Ashland, MA); incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA); cell lysis buffer and antibodies for p-MLC and caveolin-1 were from Cell Signaling Technology (Danvers, MA).

Techniques: Over Expression, Isolation, Infection, Plasmid Preparation, Immunofluorescence, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Novel Role for Non-muscle Myosin Light Chain Kinase (MLCK) in Hyperoxia-induced Recruitment of Cytoskeletal Proteins, NADPH Oxidase Activation, and Reactive Oxygen Species Generation in Lung Endothelium *

doi: 10.1074/jbc.M111.294546

Figure Lengend Snippet: Proposed model on involvement of ∼214-kDa nmMLCK in assembly and activation of non-phagocytic NADPH oxidase and ROS/superoxide production in lung endothelium. As depicted in the model, both cortactin and p47phox are diffused throughout the cell and mostly co-localized to the same intracellular compartment. Upon exposure to hyperoxia, nmMLCK facilitates the assembly and association of cortactin, p47phox, Src, and other components to the caveolin-enriched microdomains at the cell periphery for enhanced ROS/superoxide production.

Article Snippet: Antibodies for MLCK, MLC, cortactin, and p47, protein A/G plus agarose, and BSA were from Santa Cruz Biotechnology (Santa Cruz, CA); scrambled RNA and siRNA MLCK were from Dharmacon (Lafayette, CO); shMLCK-GFP was from Open Biosystems (Huntsville, AL); adenoviral construct nmMLCK-FLAG (WT) was generated at the services of the University of Iowa Gene Transfer Vector Core (Iowa City, IA); Rac1 antibody was from BD Biosciences; Gene silencer was from Genlantis (San Diego, CA); FuGENE HD transfection reagent was from Roche Applied Science; phosphatase inhibitor mixture, ML-7, and antibodies for actin, phospho-Src (Tyr-418), and FLAG were from Sigma; anti-phospho-cortactin (Tyr-486) antibody was from Chemicon (Boronia, Australia); microscopy Lab-Tek slide chambers were from Electron Microscopy Sciences (Hatfield, PA); glass bottom 35-mm dishes were from MatTek Corp. (Ashland, MA); incubator chamber for hyperoxia exposure was from Billups-Rothenberg (Del Mar, CA); cell lysis buffer and antibodies for p-MLC and caveolin-1 were from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay