Journal: Cells

Article Title: 15-Deoxy-Δ 12,14 -prostaglandin J 2 Upregulates VEGF Expression via NRF2 and Heme Oxygenase-1 in Human Breast Cancer Cells

doi: 10.3390/cells10030526

Figure Lengend Snippet: The expression levels of cyclooxygenase-2 (COX-2) and nuclear factor erythroid 2-related factor 2 (NRF2) and 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) production are increased in human breast cancer tissues. ( A ) The expression of COX-2 and NRF2 in human breast cancer tissues vs. surrounding normal tissues. Paraffin-embedded human breast cancer or adjacent normal tissues were immunostained for COX-2 and NRF2 and counterstained with hematoxylin, as described in the Materials and Methods section. Scale bars indicate 100 μm. ( B ) Formation of 15d-PGJ 2 . Phospholipase A 2 catalyzes hydrolytic release of arachidonic acid (AA) from membrane phospholipids. Cyclooxygenases catalyze oxidative conversion of arachidonic acid (AA) to PGH 2 . PGD 2 , a precursor of 15d-PGJ 2 , is formed by the action of PGD 2 synthase. Alternatively, other prostaglandins (e.g., PGE 2 , PGF 2α , and PGI 2 ) and thromboxanes are formed. PGD 2 undergoes chemical dehydration to form the PGJ 2 . PGJ 2 is then subjected to further dehydration by loss of the 15-hydroxyl group, which, coupled with migration of the 13,14-double bond of PGJ 2 , results in the formation of 15d-PGJ 2 . Asterisks indicate the positions of the chemically reactive electrophilic carbon center. ( C ) The elevated levels of 15d-PGJ 2 in human breast cancer tissues. 15d-PGJ 2 production in human breast cancer and adjacent normal tissues was measured as described in the Materials and Methods section. Data are means ± standard deviation (SD). *** p < 0.001, significantly different compared to adjacent normal breast tissues.

Article Snippet: The amounts of 15d-PGJ 2 were measured by using the 15d-PGJ 2 enzyme-immunoassay (EIA) kit (Assay Designs, Inc.; Ann Arber, MI, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Membrane, Migration, Standard Deviation

Journal: Cells

Article Title: 15-Deoxy-Δ 12,14 -prostaglandin J 2 Upregulates VEGF Expression via NRF2 and Heme Oxygenase-1 in Human Breast Cancer Cells

doi: 10.3390/cells10030526

Figure Lengend Snippet: 15d-PGJ 2 induces nuclear translocation and antioxidant responsive element (ARE) binding of NRF2. ( A ) The level of nuclear NRF2 was assessed by Western blot analysis. Data are means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001, significantly different compared to control. ( B ) Immunocytochemical analysis was performed using anti-NRF2 antibody after the treatment of MCF-7 cells with 30 µM 15d-PGJ 2 for 12 h. Cells were stained with propidium iodide (PI) and visualized by confocal microscopy. Scale bars indicate 50 μm. ( C ) Immunocytochemical detection of phosphorylated Nrf2 in MCF-7 cells with 30 µM 15d-PGJ 2 for 24 h. Cells were also stained with 4′,6-diamidino-2-phenylindole (DAPI) to identify the nuclei (blue). Scale bars indicate 200 μm. ( D ) The phosphorylation of NRF2 was assessed by Western blot analysis. The experimental conditions are same as (C). ( E ) The ARE binding activity was measured by electrophoretic mobility shift assay (EMSA). Nuclear extracts were isolated after treatment with 30 µM 15d-PGJ 2 for indicated time periods. ( F ) For the comparison of transcriptional activity of NRF2, cells were transiently transfected with plasmid (ARE-Luc) harboring the ARE-binding site-luciferase construct, GC mutant ARE (GC-MT), or control vector (Ti-Luc). After overnight transfection, cells were exposed to 30 µM 15d-PGJ 2 for 12 h and treated with reporter lysis buffer for the measurement of the luciferase activity. ** p < 0.01, significantly different from empty vector-transfected control; ## p < 0.01, significantly different compared to hARE-transfected control.

Article Snippet: The amounts of 15d-PGJ 2 were measured by using the 15d-PGJ 2 enzyme-immunoassay (EIA) kit (Assay Designs, Inc.; Ann Arber, MI, USA) according to the manufacturer’s protocol.

Techniques: Translocation Assay, Binding Assay, Western Blot, Control, Staining, Confocal Microscopy, Phospho-proteomics, Activity Assay, Electrophoretic Mobility Shift Assay, Isolation, Comparison, Transfection, Plasmid Preparation, Luciferase, Construct, Mutagenesis, Lysis

Journal: Cells

Article Title: 15-Deoxy-Δ 12,14 -prostaglandin J 2 Upregulates VEGF Expression via NRF2 and Heme Oxygenase-1 in Human Breast Cancer Cells

doi: 10.3390/cells10030526

Figure Lengend Snippet: NRF2 mediates 15d-PGJ 2 -induced vascular endothelial growth factor (VEGF) expression and angiogenesis. ( A ) The transcriptional activity of VEGF was measured by the luciferase reporter gene assay. After transfection with the VEGF-luciferase construct, MCF-7 cells were treated with 15d-PGJ 2 (30 μM) for 12 h. * p < 0.05, significantly different compared to empty vector-transfected control. ( B ) MCF-7 cells were treated with 30 µM of 15d-PGJ 2 and incubated for indicated time periods. Protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot analysis was conducted for detecting the expression of VEGF protein. Data are means ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001, significantly different compared to control. ( C ) An enzyme immunoassay was conducted to measure VEGF production by 15d-PGJ 2 in MCF-7 cells. * p < 0.05 and *** p < 0.001, significantly different from vehicle control. ( D ) Aortic segments were harvested from SD rats (n = 6 per group). Aorta in matrigel was treated with 1 μM of 15d-PGJ 2 for 5 days. Endothelial-cell sprouts forming branching cords from the margins of vessel segments taken from rat were photographed under a phase-contrast microscope. Sprouting scores were classified from 0 (least positive) to 5 (most positive). Data are means ± SD. ** p < 0.01, significantly different compared to vehicle control. ( E,F ) MCF-7 cells were transfected with NRF2 siRNA for 24 h and then incubated with 15d-PGJ 2 for an additional 24 h. The expression (E) and production (F) of VEGF were measured by Western blot analysis and enzyme-linked immunosorbent assay (ELISA), respectively. Data are means ± SD. *** p < 0.001, significantly different compared to nonspecific siRNA-transfected control. ( G ) After transfection of MCF-7 cells with control siRNA or siNRF2, the VEGF promoter activity was determined by the luciferase reporter gene assay as described in the Materials and Methods section. *** p < 0.001, significantly differrent from vehicle control; ### p < 0.001, significantly different compared to the 15d-PGJ 2 -treated control siRNA-transfected cells. ( H ) Aortic segments were harvested from NRF2 -/- KO and NRF2 +/+ wild-type (WT) mice (n = 10 per group). Aortas in Matrigel were treated with 1 μM of 15d-PGJ 2 for 5 days. Endothelial-cell sprouts forming branching cords from the margins of vessel segments taken from mice were photographed under a phase-contrast microscope. Sprouting scores were classified from 0 (least positive) to 5 (most positive). Data are means ± SD. *** p < 0.001, significantly different from vehicle control; ### p < 0.001, significantly different compared to the 15d-PGJ 2 -treated NRF2 WT group.

Article Snippet: The amounts of 15d-PGJ 2 were measured by using the 15d-PGJ 2 enzyme-immunoassay (EIA) kit (Assay Designs, Inc.; Ann Arber, MI, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Activity Assay, Luciferase, Reporter Gene Assay, Transfection, Construct, Plasmid Preparation, Control, Incubation, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Microscopy

Journal: Cells

Article Title: 15-Deoxy-Δ 12,14 -prostaglandin J 2 Upregulates VEGF Expression via NRF2 and Heme Oxygenase-1 in Human Breast Cancer Cells

doi: 10.3390/cells10030526

Figure Lengend Snippet: The α,β-unsaturated carbonyl moiety of 15d-PGJ 2 is essential for the activation of NRF2, expression/production of VEGF, and further angiogenesis induced by this cyclopentenone prostaglandin. ( A ) MCF-7 cells were incubated with 30 μM of biotinylated 15d-PGJ 2 in the presence of dithiothreitiol (DTT) (1 mM) and N-acetylcysteine (NAC) (5 mM) for 9 h. The interaction between Keap1 and 15d-PGJ 2 was assessed by immunoblot analysis, and the incorporation of biotinylated 15d-PGJ 2 into Keap1 immunoprecipitates was detected with horseradish peroxidase (HRP)-streptavidin and enhanced chemiluminescence (ECL). ( B ) The chemical structures of 15d-PGJ 2 and biotinylated 9,10-dihydro-15d-PGJ 2 (dh-PGJ 2 ). Asterisks depict electrophilic carbons (position 9 and 13). ( C ) MCF-7 cells were incubated with 30 μM of biotinylated 15d-PGJ 2 or biotinylated dh-PGJ 2 . The interaction between Keap1 and 15d-PGJ 2 or dh-PGJ 2 was assessed by immunoblot analysis with HRP-streptavidin and ECL. ( D ) Immunocytochemical analysis was performed using anti-NRF2 antibody after the treatment of MCF-7 cells with 30 µM each of 15d-PGJ 2 and dh-PGJ 2 for 12 h. Scale bars indicate 50 μm. Western blot analysis ( E ) and ELISA ( F ) and were performed to examine the levels of VEGF in MCF-7 cell treated with 15d-PGJ 2 or dh-PGJ 2 . Data are means ± SD. *** p < 0.001, significantly different compared to vehicle control; # p < 0.05 and ### p < 0.001, significantly different compared to 15d-PGJ 2 -treated cells. ( G ) HUVECs were treated with various conditioned media (CM) with 10% fetal bovine serum (FBS). After 16 h incubation with 15d-PGJ 2 and dh-PGJ 2 , microphotographs were taken 40×. Representative endothelial tubes were shown. Tube formation (%) was quantified by the number of three-way branching point. Values are means ± SD of three independent experiments. *** p < 0.001, significantly different from vehicle control; ### p < 0.001, significantly different compared to 15d-PGJ 2 -treated cells.

Article Snippet: The amounts of 15d-PGJ 2 were measured by using the 15d-PGJ 2 enzyme-immunoassay (EIA) kit (Assay Designs, Inc.; Ann Arber, MI, USA) according to the manufacturer’s protocol.

Techniques: Activation Assay, Expressing, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Cells

Article Title: 15-Deoxy-Δ 12,14 -prostaglandin J 2 Upregulates VEGF Expression via NRF2 and Heme Oxygenase-1 in Human Breast Cancer Cells

doi: 10.3390/cells10030526

Figure Lengend Snippet: 15d-PGJ 2 induces expression/activity of HO-1 through NRF2 activation. ( A ) Cluster analysis of microarray data. Hierarchical clustering of 12 samples and 16,694 probes sets with significant variation in RNA expression levels across all samples. Overall analysis of 12 experiments carried out for microarray analysis of the gene expression profiles in MCF-7 cells after treatment with 30 μM each of 15d-PGJ 2 or 9,10-dihydro-15d-PGJ 2 (dh-PGJ 2 ) for 12 h. Downregulated genes are represented in green (Cy3) and upregulated genes in red (Cy5). The horizontal color bar provides a visual indication of the level of changes in each PGJ 2 -treated cell vs. control. ( B ) Western blot analysis was performed to examine the expression of HO-1 protein in MCF-7 cell treated with 15d-PGJ 2 (30 μM) or dh-PGJ 2 (30 μM). ( C ) RT-PCR was conducted to examine the levels of the HO-1 mRNA transcript in MCF-7 cell treated with 15d-PGJ 2 (0, 3, and 30 μM). (D ) Immunocytochemical localization of HO-1 was analyzed after the treatment of MCF-7 cells with 30 µM 15d-PGJ 2 for 24 h. Cells were also stained with DAPI to visualize the nuclei (blue). Scale bars indicate 200 μm. ( E ) MCF-7 cells were treated with 15d-PGJ 2 for 12 h and harvested to determine the ARE binding activity by the chromatin immunoprecipitation (ChIP) assay. Chromatin immunoprecipitated DNA was analyzed by RT-PCR with primers for ARE element-containing region of the human HO-1 promoter. ( F ) MCF-7 cells were transfected with control or NRF2 siRNA (1, 5, and 10 μM) for 24 h and then incubated with 30 μM 15d-PGJ 2 for an additional 24 h. The expression of HO-1 was measured by Western blotting. ( G ) MCF-7 cells were transfected with control vector (pEF) and DN-NRF2. After 24 h of incubation, cells were treated with 30 µM of 15d-PGJ 2 for 24 h. The expression of NRF2 and HO-1 was determined by Western blot analysis. Data are means ± SD. ** p < 0.01, significantly different compared to vehicle control; ## p < 0.01, significantly different from pEF-transfected cells. ( H ) 15d-PGJ 2 -induced HO activity was determined in MCF-7 cells transfected with DN-NRF2 as well as in vector-transfected control cells. Data are means ± SD. * p < 0.05, significantly different compared to vehicle control; # p < 0.05, significantly different from empty vector-transfected cells in the presence of 15d-PGJ 2 .

Article Snippet: The amounts of 15d-PGJ 2 were measured by using the 15d-PGJ 2 enzyme-immunoassay (EIA) kit (Assay Designs, Inc.; Ann Arber, MI, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Activity Assay, Activation Assay, Microarray, RNA Expression, Gene Expression, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Staining, Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Transfection, Incubation, Plasmid Preparation

Journal: Cells

Article Title: 15-Deoxy-Δ 12,14 -prostaglandin J 2 Upregulates VEGF Expression via NRF2 and Heme Oxygenase-1 in Human Breast Cancer Cells

doi: 10.3390/cells10030526

Figure Lengend Snippet: 15d-PGJ 2 induces angiogenesis through upregulation of HO-1 mediated by NRF2 activation. ( A ) 15d-PGJ 2 -induced HO activity was determined in MCF-7 cells treated with zinc protoporphyrin IX (ZnPP) (10 μM), an HO-1 inhibitor, for 24 h. Data are means ± SD. ** p < 0.01, significantly different compared to vehicle control; ## p < 0.01, significantly different from 15d-PGJ 2 -treated cells. ( B ) Treatment with ZnPP (1 5, and 20 μM) abrogated the 15d-PGJ 2 -induced expression of VEGF protein and mRNA. Data are means ± SD. ** p < 0.01 and *** p < 0.001, significantly different compared to vehicle control; # p < 0.05 and ## p < 0.01, significantly different from 15d-PGJ 2 -treated cells. ( C ) The enzyme immunoassay was conducted to measure the VEGF production in MCF-7 cells exposed to 30 µM 15d-PGJ 2 in the presence or absence of ZnPP. Data are means ± SD. *** p < 0.001, significantly different compared to vehicle control; ### p < 0.001, significantly different from 15d-PGJ 2 -treated cells. ( D ) MCF-7 cells were transfected with HO-1 siRNA for 24 h and treated with 15d-PGJ 2 for an additional 24 h. Western blotting was conducted to confirm HO-1 knockdown. The production of VEGF was determined by ELISA. Data are means ± SD. *** p < 0.001, significantly different compared to nonspecific siRNA-transfected cells. ( E ) HUVECs were treated with conditioned media containing 10% FBS. After 16 h incubation with 15d-PGJ 2 (0 or 30 µM) in the presence or absence of 10 µM ZnPP, microphotographs were taken (’40). Representative endothelial tubes were shown. Tube formation (%) was quantified by the number of three-way branching point. Values are means ± SD of three independent experiments. *** p < 0.001, significantly different compared to vehicle control; ### p < 0.001, significantly different from 15d-PGJ 2 -treated cells.

Article Snippet: The amounts of 15d-PGJ 2 were measured by using the 15d-PGJ 2 enzyme-immunoassay (EIA) kit (Assay Designs, Inc.; Ann Arber, MI, USA) according to the manufacturer’s protocol.

Techniques: Activation Assay, Activity Assay, Control, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Knockdown, Incubation

Journal: Molecular and Cellular Biology

Article Title: Transcription Factor Nrf2 Regulates Inflammation by Mediating the Effect of 15-Deoxy-Δ 12,14 -Prostaglandin J 2

doi: 10.1128/mcb.24.1.36-45.2004

Figure Lengend Snippet: FIG. 2. Accumulation of 15d-PGJ2 (brown) in pleural macro- phages during carrageenan-induced pleurisy. Pleural inflammatory cells were examined immunohistochemically at the indicated times of pleurisy with anti-15d-PGJ2 antibody. Arrows indicate macrophages, while arrowheads indicate neutrophils.

Article Snippet: The sections were reacted with anti-15d-PGJ2 monoclonal antibody (38), anti-PrxI antibody (16), anti-HO-1 antibody (a generous gift from Shigeru Taketani), anti-F4/80 antibody (Serotec), anti-COX-2 antibody (Santa Cruz), or anti-hematopoietic PG synthetase (PGDS) antibody (Santa Cruz) and incubated for another hour with Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Transcription Factor Nrf2 Regulates Inflammation by Mediating the Effect of 15-Deoxy-Δ 12,14 -Prostaglandin J 2

doi: 10.1128/mcb.24.1.36-45.2004

Figure Lengend Snippet: FIG. 3. cyPGs activate a set of antioxidant and anti-inflammatory genes in mouse peritoneal macrophages. (A) Peritoneal macrophages were treated with dimethyl sulfoxide alone (lane 1); with 15d-PGJ2 at 0.5 M (lane 2), 1 M (lane 3), 5 M (lane 4), or 10 M (lane 5) for 5 h; or with 100 M diethylmaleate for 5 h (lane 6), and total RNAs were analyzed by RNA blot analysis with HO-1, PrxI, and A170 cD- NAs as probes. A -actin cDNA probe was used for a loading control. (B) Peritoneal macrophages were treated with dimethyl sulfoxide alone (lane 1), PGA1 (50 M) (lane 2), PGB2 (50 M) (lane 3), PGD2 (50 M) (lane 4), PGE2 (50 M) (lane 5), PGF1a (50 M) (lane 6), 15d-PGJ2 (10 M) (lane 7), thromboxane B2 (50 M) (lane 8), leu- kotriene B4 (1 M) (lane 9), arachidonic acid (50 M) (lane 10), or diethylmaleate (100 M) (lane 11). Total RNA fractions were ana- lyzed by RNA blot analysis as for panel A.

Article Snippet: The sections were reacted with anti-15d-PGJ2 monoclonal antibody (38), anti-PrxI antibody (16), anti-HO-1 antibody (a generous gift from Shigeru Taketani), anti-F4/80 antibody (Serotec), anti-COX-2 antibody (Santa Cruz), or anti-hematopoietic PG synthetase (PGDS) antibody (Santa Cruz) and incubated for another hour with Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan).

Techniques: Northern blot, Control

Journal: Molecular and Cellular Biology

Article Title: Transcription Factor Nrf2 Regulates Inflammation by Mediating the Effect of 15-Deoxy-Δ 12,14 -Prostaglandin J 2

doi: 10.1128/mcb.24.1.36-45.2004

Figure Lengend Snippet: FIG. 4. cyPGs activate Nrf2 in macrophages and hepatocytes. (A) Peritoneal macrophages derived from either wild-type mice (lanes 1 to 4) or nrf2/ mice (lanes 5 to 8) were treated with 5 M (lanes 2 and 6) or 10 M (lanes 3 and 7) 15d-PGJ2 or with 100 M diethylmaleate (lanes 4 and 8). Untreated controls are shown in lanes 1 and 5. Total RNAs were analyzed by RNA blotting analysis, using cDNAs for HO-1, PrxI, or A170 as probes. A -actin cDNA probe was used for a loading control. (B) Nuclear extracts were prepared from macrophages untreated (lane 1) or treated with diethylmaleate (100 M) (lane 2), 15d-PGJ2 (5 M) (lane 3), PGA1 (100 M) (lane 4), or PGE2 (100 M) (lane 5). The extracts were immunoblotted with anti-Nrf2 antibody or anti-lamin B antibody. Recombinant Nrf2 in 293T cells was also loaded as a control (lane 6). (C and D) RL34 cells were either untreated (C) or treated with 10 M 15d-PGJ2 for 4 h (D), and expression of Nrf2 were examined immunocytochem- ically with the anti-Nrf2 antibody. Subcellular localization of Nrf2 was analyzed by confocal microscopy. (E) RL34 cells were treated with dimethyl sulfoxide (lane 1), biotinylated 12-PGJ2 (lane 2), or biotinylated 15d-PGJ2 (lane 3). Keap1-PGJ2 complexes were precipitated from the cell extracts with avidin beads and probed with the anti-Keap1 antibody.

Article Snippet: The sections were reacted with anti-15d-PGJ2 monoclonal antibody (38), anti-PrxI antibody (16), anti-HO-1 antibody (a generous gift from Shigeru Taketani), anti-F4/80 antibody (Serotec), anti-COX-2 antibody (Santa Cruz), or anti-hematopoietic PG synthetase (PGDS) antibody (Santa Cruz) and incubated for another hour with Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan).

Techniques: Derivative Assay, Control, Recombinant, Expressing, Confocal Microscopy, Avidin-Biotin Assay

Journal: Molecular and Cellular Biology

Article Title: Transcription Factor Nrf2 Regulates Inflammation by Mediating the Effect of 15-Deoxy-Δ 12,14 -Prostaglandin J 2

doi: 10.1128/mcb.24.1.36-45.2004

Figure Lengend Snippet: FIG. 5. PrxI expression at the early phase of carrageenan-induced pleurisy depends on both 15d-PGJ2 and Nrf2. (A) Both the COX-2 inhibitor NS-398 and nrf2 gene disruption affect PrxI expression during carrageenan-induced pleurisy. Whole-cell extracts of pleural inflammatory cells from wild-type mice (lanes 1, 3, 6, 9, 12, and 15), nrf2/ mice (lanes 2, 4, 7, 10, 13, and 16), or NS-398-treated wild-type mice (lanes 5, 8, 11, 14, and 17) at the indicated time points of pleurisy were examined by immunoblotting with anti-PrxI antibody. (B) The expression of PrxI, 15d-PGJ2, F4/80, HO-1, COX-2, and hematopoietic PGDS in pleural inflammatory cells at 24 h of pleurisy was analyzed immunohistochemically with the corresponding antibodies as indicated. Arrows indicate macrophages, while arrowheads represent neutrophils. (C and D) Administration of NS-398 affects neutrophil (C) and macrophage (D) numbers during carrageenan-induced pleurisy. The number of pleural inflammatory cells was microscopically counted, and the means from four experiments are presented with standard errors of the mean. *, significantly different from time-matched NS398-untreated mice (P 0.05). a, significantly different from carrageenan-untreated wild-type mice at the 0-h time point (P 0.05). b, significantly different from carrageenan-untreated nrf2/ mice at the 0-h time point (P 0.05). (E) Effect of indomethacin on pleural inflammatory cells. Cell counts were taken at 24 h of pleurisy. The means from four experiments are presented with standard errors of the mean. *, significantly different from untreated control mice (P 0.05).

Article Snippet: The sections were reacted with anti-15d-PGJ2 monoclonal antibody (38), anti-PrxI antibody (16), anti-HO-1 antibody (a generous gift from Shigeru Taketani), anti-F4/80 antibody (Serotec), anti-COX-2 antibody (Santa Cruz), or anti-hematopoietic PG synthetase (PGDS) antibody (Santa Cruz) and incubated for another hour with Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan).

Techniques: Expressing, Disruption, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: Transcription Factor Nrf2 Regulates Inflammation by Mediating the Effect of 15-Deoxy-Δ 12,14 -Prostaglandin J 2

doi: 10.1128/mcb.24.1.36-45.2004

Figure Lengend Snippet: FIG. 6. Intrapleural injection of 15d-PGJ2 reverses the decrease of PrxI expression and persistence of neutrophils in NS-398-treated mice but not in Nrf2-deficient mice. (A and B) Numbers of neutrophils (A) and macrophages (B) at 24 h of pleurisy in wild-type (nrf2/) mice (bars 1), NS-398-treated nrf2/ mice (bars 2), NS-398-treated nrf2/ mice administrated 15d-PGJ2 (bars 3), nrf2/ mice (bars 4), and nrf2/ mice administrated NS-398 (bars 5). Neutrophils and macrophages were counted microscopically, and the means and stan- dard deviations of triplicates are shown. a, P 0.05 compared with untreated control mice; b, P 0.01 compared with untreated control mice; c, P 0.01 compared with NS-398-treated wild-type mice. (C, D) RT-PCR analysis of PrxI (C) and HO-1 (D) mRNAs in pleural in- flammatory cells in mice. Intensities of RT-PCR bands were quantified by densitometric analysis, and the means of triplicates are presented. a, P 0.01 compared with untreated control group; b, P 0.01 com- pared with NS-398-treated wild-type mice.

Article Snippet: The sections were reacted with anti-15d-PGJ2 monoclonal antibody (38), anti-PrxI antibody (16), anti-HO-1 antibody (a generous gift from Shigeru Taketani), anti-F4/80 antibody (Serotec), anti-COX-2 antibody (Santa Cruz), or anti-hematopoietic PG synthetase (PGDS) antibody (Santa Cruz) and incubated for another hour with Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan).

Techniques: Injection, Expressing, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular and Cellular Biology

Article Title: Transcription Factor Nrf2 Regulates Inflammation by Mediating the Effect of 15-Deoxy-Δ 12,14 -Prostaglandin J 2

doi: 10.1128/mcb.24.1.36-45.2004

Figure Lengend Snippet: FIG. 7. Network of 15d-PGJ2 and the Nrf2-Keap1 pathway in car- rageenan-induced pleurisy. 15d-PGJ2 is generated in pleural macro- phages during carrageenan-induced pleurisy and is known to directly inhibit NF-B activity and also to act as a ligand for PPAR. This study shows that 15d-PGJ2 activates the Nrf2-Keap1 pathway through cova- lent binding to Keap1. Nrf2 induces HO-1 and PrxI expression as well as other ARE-regulated genes in macrophages (green). The upregu- lation of PrxI appears to inhibit NF-B, TNF- , and the -tautomerase activity of MIF, thus modulating the inflammation process. On the other hand, HO-1 is known to inhibit the expression of TNF- through CO production.

Article Snippet: The sections were reacted with anti-15d-PGJ2 monoclonal antibody (38), anti-PrxI antibody (16), anti-HO-1 antibody (a generous gift from Shigeru Taketani), anti-F4/80 antibody (Serotec), anti-COX-2 antibody (Santa Cruz), or anti-hematopoietic PG synthetase (PGDS) antibody (Santa Cruz) and incubated for another hour with Histofine Simple Stain MAX-PO (Nichirei, Tokyo, Japan).

Techniques: Generated, Activity Assay, Binding Assay, Expressing

Journal: Neurotoxicity Research

Article Title: Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ 2 Attenuates H 2 O 2 -Induced IL-6 Expression in Rat Brain Astrocytes

doi: 10.1007/s12640-020-00318-6

Figure Lengend Snippet: 15d-PGJ 2 induced NRROS expression in RBA-1 cells. a , b RBA-1 cells were treated with 1, 5, or 10 μM 15d-PGJ 2 for the indicated time intervals. The levels of a NRROS protein and b mRNA expression were analyzed by Western blot and RT/real-time PCR, respectively. Lower panel of a indicates the quantitation of 10 μM 15d-PGJ 2 treatment. c RBA-1 cells were pretreated with actinomycin D (ActD, 1 μM), mithramycin A (Mith A, 1 μM), GW9662 (GW, 10 nM), LY294002 (10 μM), Akti VIII (1 μM), or AS1842856 (AS, 0.3 μM) for 1 h and then treated with 10 μM 15d-PGJ 2 for 2 h. The levels of NRROS mRNA expression were analyzed by RT/real-time PCR. The data are expressed as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significant difference as indicated

Article Snippet: 15d-PGJ 2 and 15d-PGJ 2 antibody (Cat# ADI-915-043-100) were from Enzo (New York, NY, USA).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitation Assay, Cell Culture

Journal: Neurotoxicity Research

Article Title: Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ 2 Attenuates H 2 O 2 -Induced IL-6 Expression in Rat Brain Astrocytes

doi: 10.1007/s12640-020-00318-6

Figure Lengend Snippet: Analysis of rat NRTROS promoter and screening for 15d-PGJ 2 response elements. a The DNA sequences of rat NRROS promoter and transcription factor binding sites. The exon 1 regions are shown in boldface and (+ 1) indicates the first nucleotide of the mRNA. b RBA-1 cells were transfected with either rat (r)NRROS or (h)NRROS plasmid, and pCMV-β-gal DNA for 24 h, and then treated with 10 μM 15d-PGJ 2 for the indicated time intervals. The promoter activity was determined in the cell lysates using a promoter assay kit. c Scheme of the rat NRROS promoter region and reporter constructs. The position of deletion mutant, transcription initiation (+ 1), and location of exon 1 are indicated. Figures were not drawn to scale. d RBA-1 cells transfected with various NRROS constructs were treated with 10 μM 15d-PGJ 2 for 1 h. The pGL3B-Luc without the promoter was used as a control (Basic). Deletion mutants were used to analyze the essential promoter regions and 15d-PGJ 2 response element. RLU indicates the related luciferase units. Statistical analysis was determined using two-tailed unpaired Student’s t test. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Article Snippet: 15d-PGJ 2 and 15d-PGJ 2 antibody (Cat# ADI-915-043-100) were from Enzo (New York, NY, USA).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Activity Assay, Promoter Assay, Construct, Mutagenesis, Control, Luciferase, Two Tailed Test, Cell Culture

Journal: Neurotoxicity Research

Article Title: Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ 2 Attenuates H 2 O 2 -Induced IL-6 Expression in Rat Brain Astrocytes

doi: 10.1007/s12640-020-00318-6

Figure Lengend Snippet: 15d-PGJ 2 -induced NRROS expression is mediated through PI3K, AKT, and FoxO1. RBA-1 cells were pretreated with different concentrations of a , d LY294002, b , e AKTi VIII, or c , f AS1842856 for 1 h and then incubated with 10 μM 15d-PGJ 2 for the indicated time intervals. The levels of NRROS, phospho-AKT, phospho-FoxO1, AKT, FoxO1, and GAPDH protein were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Article Snippet: 15d-PGJ 2 and 15d-PGJ 2 antibody (Cat# ADI-915-043-100) were from Enzo (New York, NY, USA).

Techniques: Expressing, Incubation, Western Blot, Cell Culture

Journal: Neurotoxicity Research

Article Title: Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ 2 Attenuates H 2 O 2 -Induced IL-6 Expression in Rat Brain Astrocytes

doi: 10.1007/s12640-020-00318-6

Figure Lengend Snippet: The roles of PI3K, AKT, and FoxO1 in 15d-PGJ 2 -induced NRROS expression are confirmed by transfection with respective siRNAs. RBA-1 cells were transfected with siRNA ( a , d p85; b , e AKT, or c , f FoxO1) or scramble siRNA for 48 h and then incubated with 10 μM 15d-PGJ 2 for 4 h ( a – c ) and for the indicated time intervals ( d – f ). The levels of NRROS, phospho-AKT, phospho-FoxO1, AKT, FoxO1, and GAPDH protein were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Article Snippet: 15d-PGJ 2 and 15d-PGJ 2 antibody (Cat# ADI-915-043-100) were from Enzo (New York, NY, USA).

Techniques: Expressing, Transfection, Incubation, Western Blot, Cell Culture

Journal: Neurotoxicity Research

Article Title: Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ 2 Attenuates H 2 O 2 -Induced IL-6 Expression in Rat Brain Astrocytes

doi: 10.1007/s12640-020-00318-6

Figure Lengend Snippet: 15d-PGJ 2 stimulates the interaction between Sp1 and FoxO1 leading to NRROS expression. a RBA-1 cells were transfected with Sp1 siRNA and then incubated with 10 μM 15d-PGJ 2 for 4 h. The levels of NRROS, Sp1, and GAPDH were analyzed by Western blotting. b Schematic representation of rat NRROS promoter. Locations of primers used for ChIP assays (Sp1 and FoxO1) and transcription factors associated regions are indicated. c , d ChIP assays were performed using an anti-Sp1 or anti-FoxO1 antibody and then amplified the DNA fragments with Sp1 and FoxO1 primers, respectively. e , f RBA-1 cells were pretreated without or with mithramycin A or AS1842856 for 1 h and then incubated with 15d-PGJ 2 for 1 h. The association between FoxO1 and Sp1 on promoter was determined by ChIP assays. g Cells were treated with 10 μM 15d-PGJ 2 for the indicated time intervals. ChIP assays were performed using an anti-FoxO1 or anti-phospho-FoxO1 antibody. h Cells were treated with 10 μM 15d-PGJ 2 for the indicated time intervals. The levels of protein expression in either cytoplasmic or nuclear fractions were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Article Snippet: 15d-PGJ 2 and 15d-PGJ 2 antibody (Cat# ADI-915-043-100) were from Enzo (New York, NY, USA).

Techniques: Expressing, Transfection, Incubation, Western Blot, Amplification, Cell Culture

Journal: Neurotoxicity Research

Article Title: Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ 2 Attenuates H 2 O 2 -Induced IL-6 Expression in Rat Brain Astrocytes

doi: 10.1007/s12640-020-00318-6

Figure Lengend Snippet: NRROS expression via Nrf2 activation by 15d-PGJ 2 in RBA-1 cells. The cells were transfected with Nrf2 siRNA or scramble siRNA ( a , b ) for 48 h and then incubated with 10 μM 15d-PGJ 2 for 4 h or 6 h ( a ), and indicated time intervals ( b ). c The cells were pretreated with various concentration of anti-15d-PGJ2 antibody for 1 h and then incubated with 10 μM 15d-PGJ 2 for 6 h. The levels of NRROS, phospho-Nrf2, Nrf2, β-actin, and GAPDH protein were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Article Snippet: 15d-PGJ 2 and 15d-PGJ 2 antibody (Cat# ADI-915-043-100) were from Enzo (New York, NY, USA).

Techniques: Expressing, Activation Assay, Transfection, Incubation, Concentration Assay, Western Blot, Cell Culture

Journal: Neurotoxicity Research

Article Title: Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ 2 Attenuates H 2 O 2 -Induced IL-6 Expression in Rat Brain Astrocytes

doi: 10.1007/s12640-020-00318-6

Figure Lengend Snippet: Overexpression of human NRROS reduces the H 2 O 2 -induced ROS generation, IL-6 expression, and p47 phosphorylation. a RBA-1 cells were pretreated with 10 μM 15d-PGJ 2 for 1 h and then treated with 100 μM H 2 O 2 for the indicated time intervals. The levels of protein expression were analyzed by Western blotting. b RBA-1 cells were pretreated with 15d-PGJ 2 for 1 h and then treated with 100 μM H 2 O 2 for the indicated time intervals. The levels of IL-6 mRNA expression were determined by RT/real-time PCR. c , d RBA-1 stable clones of hNRROS and pCMV-Tag2B, or RBA-1 cells were treated with 100 μM H 2 O 2 for 1 h. c The levels of hNRROS mRNA expression were determined by RT/real-time PCR. d The cells were labeled with 10 μM H 2 DCFDA and then incubated with 100 μM H 2 O 2 for 30 min. The levels of ROS generation were observed using a fluorescence microscope. Scale bar = 100 μm. e RBA-1 stable clones of hNRROS and pCMV-Tag2B, or RBA-1 cells were treated with 100 μM H 2 O 2 for the indicated time intervals. The levels of protein expression were analyzed by Western blotting. f , g RBA-1 stable clones were treated with H 2 O 2 for the indicated time intervals. The levels of IL-6 mRNA expression and secretion of IL-6 were analyzed by RT/real-time PCR and ELISA kit, respectively. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). * p < 0.05, as compared with the respective values significantly different as indicated

Article Snippet: 15d-PGJ 2 and 15d-PGJ 2 antibody (Cat# ADI-915-043-100) were from Enzo (New York, NY, USA).

Techniques: Over Expression, Expressing, Phospho-proteomics, Western Blot, Real-time Polymerase Chain Reaction, Clone Assay, Labeling, Incubation, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Neurotoxicity Research

Article Title: Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ 2 Attenuates H 2 O 2 -Induced IL-6 Expression in Rat Brain Astrocytes

doi: 10.1007/s12640-020-00318-6

Figure Lengend Snippet: Scheme of signaling pathways is involved in 15d-PGJ 2 -induced NRROS expression suppressing the H 2 O 2 -induced IL-6 expression and ROS generation in RBA-1 cells. NRROS expression induced by 15d-PGJ 2 is mediated through a PPARγ-independent activation of PI3K/AKT leading to phosphorylation of FoxO1 and Sp1. These activated transcription factors bind to NRROS promoters and enhance NRROS transcription in RBA-1 cells. Up-regulation of NRROS not only downregulates the H 2 O 2 -mediated ROS generation but also inhibits the expression of pro-inflammatory cytokine IL-6. These results elucidate the mechanisms underlying 15d-PGJ 2 -induced NRROS expression which might be a potential strategy for management of brain inflammatory and neurodegenerative diseases

Article Snippet: 15d-PGJ 2 and 15d-PGJ 2 antibody (Cat# ADI-915-043-100) were from Enzo (New York, NY, USA).

Techniques: Protein-Protein interactions, Expressing, Activation Assay, Phospho-proteomics