Journal: Marine Drugs

Article Title: Dual Biological Functions of a Cytoprotective Effect and Apoptosis Induction by Bioavailable Marine Carotenoid Fucoxanthinol through Modulation of the Nrf2 Activation in RAW264.7 Macrophage Cells

doi: 10.3390/md15100305

Figure Lengend Snippet: Expression of HO-1 expression due to marine carotenoid, fucoxanthin (Fx) and its metabolite fucoxanthinol (FxOH) in RAW 264.7 macrophage cells. The HO-1 protein expression due to marine carotenoids (2.5–10 μM), the Fx and its metabolite FxOH effect on the Nrf2-ARE signaling in the cells was examined. ( a ) Western blot analysis of the HO-1 protein in the presence of compounds. ( b ) Densitometry analysis of the expression of HO-1 protein. Data were expressed as mean ( n = 3) ± SD. Analysis of data was carried out using ANOVA and Tukey’s method for multiple comparisons. A significance p < 0.05 between two bars with the different alphabetical letters was considered statistically significant.

Article Snippet: The products of anti-Nrf2 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-HO-1 (StressMarq Biosciences Inc., Victoria, BC, Canada), anti-Bcl-xL (BioVision, Inc., Milpitas, CA, USA), anti-phospho Akt (Cell Signaling Technology Japan, Inc., Tokyo, Japan), and anti-β-actin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were used for detecting the protein expression.

Techniques: Expressing, Western Blot

Journal: Blood

Article Title: Wild-type macrophages reverse disease in heme oxygenase 1-deficient mice

doi: 10.1182/blood-2014-02-554162

Figure Lengend Snippet: Nonmyeloablative BM transplantation in Hmox1−/− mice improved blood chemistries and led to resolution of anemia. (A) Scheme of the BM transplantation experiment used for subsequent data collection. (B) BM engraftment dynamics are represented as a percentage of CD45.2-positive leukocytes in peripheral blood estimated by fluorescence-activated cell sorter analysis. Changes over weeks are plotted for each individual mouse. (C) Hmox1−/− mice had elevated serum ALP and LDH levels. The blood chemistries returned to normal in BM transplanted recipients. (D) Indicators of microcytic anemia mean cell volume and hematocrit improved in BM-transplanted Hmox1−/− mice. (C-D) Average values for the terminal time, 21 weeks, are shown for each experimental group; error bars represent the standard deviation (N = 5). TMS water, drinking water supplemented with trimethoprim (300 μg/ml) and sulfamethoxazole (60 μg/ml).

Article Snippet: Human liver tissue slides were treated with appropriate dilutions of rabbit anti-HMOX1 (SPC-112 C/D; StressMark Biosciences, Inc.) or mouse monoclonal anti-CD163 (CM353; Biocare Medical).

Techniques: Transplantation Assay, Fluorescence, Standard Deviation

Journal: Blood

Article Title: Wild-type macrophages reverse disease in heme oxygenase 1-deficient mice

doi: 10.1182/blood-2014-02-554162

Figure Lengend Snippet: BMT reduced the oxidative stress response and prevented injury to Hmox1−/− kidneys. mRNA levels of oxidative stress responsive genes were significantly elevated in KO Ctr animals, including Gsta2 (glutamine-S-transferase A2) (A), Gstm1 (glutamine-S-transferase μ 1) (B), Gclc (glutamate-cysteine ligase, catalytic subunit) (C), Nqo1 [NAD(P)H:quinone dehydrogenase, quinone 1] (D), Fpn1 (ferroportin 1) (E), and Mrp2 (F), but returned to normal after BMT. (G) Masson’s trichrome staining of paraffin-embedded kidney tissues revealed significant accumulation of collagen, which is colored blue (arrows) in KO Ctr mice. Collagen depositions were minimal in transplanted animals. Scale bar represents 50 μm.

Article Snippet: Human liver tissue slides were treated with appropriate dilutions of rabbit anti-HMOX1 (SPC-112 C/D; StressMark Biosciences, Inc.) or mouse monoclonal anti-CD163 (CM353; Biocare Medical).

Techniques: Staining

Journal: Blood

Article Title: Wild-type macrophages reverse disease in heme oxygenase 1-deficient mice

doi: 10.1182/blood-2014-02-554162

Figure Lengend Snippet: Hmox1 mRNA expression and quantification of Hmox1+/+ DNA in transplanted Hmox1−/− animals. Hmox1 mRNA expression levels in the tissues of KO BMT animals were partially restored to normal in the BM (A) and spleen (B), were twofold higher in the liver (C), and were negligibly low in the kidney (D) in comparison with WT animals. Data were obtained by quantitative reverse-transcription polymerase chain reaction. ND, not detected. (E) Percentage of WT cells in the tissues of KO BMT animals, as assessed by quantification of the WT Hmox1 gene in a total genomic DNA extracts; we observed that liver had the highest number of donor cells of the 3 key tissues analyzed, at 3%. Primers that target exon 3 of the Hmox1 gene, which was deleted in Hmox1−/− mice, were used for the quantification.

Article Snippet: Human liver tissue slides were treated with appropriate dilutions of rabbit anti-HMOX1 (SPC-112 C/D; StressMark Biosciences, Inc.) or mouse monoclonal anti-CD163 (CM353; Biocare Medical).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Blood

Article Title: Wild-type macrophages reverse disease in heme oxygenase 1-deficient mice

doi: 10.1182/blood-2014-02-554162

Figure Lengend Snippet: Comparison of Hmox1 protein expression in mouse and human liver tissue of controls with Hmox1-deficient animals and patient samples. (A) Cross-sections of paraffin-embedded liver tissue immunofluorescence showed that Kupffer cells were a major site of Hmox1 expression in both WT Ctr and WT BMT mice (left); no specific Hmox1 signal was detectable in the liver KO Ctr animals (upper right); an Hmox1-expressing Kupffer cell population was restored in the liver of transplant recipients (bottom right). Arrows point to Hmox1-positive cells. (B) A human liver biopsy specimen obtained from human with intact HMOX1 gene showed high HMOX1 signal present in Kupffer-like cells (left), whereas there was no HMOX1 expression in the liver sample of an HMOX1-deficient patient specimen (right). Scale bars represent 50 μm (A) and 25 μm (B).

Article Snippet: Human liver tissue slides were treated with appropriate dilutions of rabbit anti-HMOX1 (SPC-112 C/D; StressMark Biosciences, Inc.) or mouse monoclonal anti-CD163 (CM353; Biocare Medical).

Techniques: Expressing, Immunofluorescence

Journal: Blood

Article Title: Wild-type macrophages reverse disease in heme oxygenase 1-deficient mice

doi: 10.1182/blood-2014-02-554162

Figure Lengend Snippet: Kupffer cells are absent in the livers of Hmox1−/− animals and a human patient, but BMT restores Kupffer cells to Hmox1−/− mice. (A) Immunohistochemistry for the pan-macrophage marker, F4/80, indicated that Kupffer cells were virtually absent in the livers of Hmox1−/− sham mice (upper middle and right) in comparison with WT mice (left). F4/80-positive cells turned brown after diaminobenzidine staining (arrowheads). BMT completely restored macrophage populations in Hmox1−/− animals (lower middle and right). Results are shown for Hmox1−/− animals that were 4 months (middle) or 1.7 months (right) old at the time the BMT procedure was performed. (B) Expression of the marker of M2 polarized macrophages indicated that CD163 was partially restored in liver, BM, and spleen of KO BMT mice, as determined by quantitative reverse-transcription polymerase chain reaction. ND, not detected. (C) CD163 Kupffer cells normally found in human liver (left) were undetectable in the liver of an HMOX1-deficient patient (right). Scale bars represent 50 μm (A) and 25 μm (C).

Article Snippet: Human liver tissue slides were treated with appropriate dilutions of rabbit anti-HMOX1 (SPC-112 C/D; StressMark Biosciences, Inc.) or mouse monoclonal anti-CD163 (CM353; Biocare Medical).

Techniques: Immunohistochemistry, Marker, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Antioxidants

Article Title: Induction of BVR-A Expression by Korean Red Ginseng in Murine Hippocampal Astrocytes: Role of Bilirubin in Mitochondrial Function via the LKB1–SIRT1–ERRα Axis

doi: 10.3390/antiox11091742

Figure Lengend Snippet: KRGE induces BVR-A and GFAP co-expression in the fimbria region of the mouse hippocampus. ( a ) Representative images of BVR-A (green) and GFAP (red) staining of mouse brains exposed to water or 0.25 mg/mL KRGE for 3 days ( n = 3 per group). Dotted lines indicate the area of hippocampal fimbria. Scale bar = 100 µm. ( b ) Co-localized staining can be observed as yellow color in the fimbria region of 0.25 mg/mL KRGE-treated mice. Scale bars = 50 µm. ( c ) The expression levels of BVR-A and β-actin proteins in the brain tissues were determined using Western blot analysis, and BVR-A levels were quantified ( n = 4 per group). ( d ) Astrocytes were incubated with KRGE at various concentrations for 24 h, and BVR-A levels were detected by Western blot analysis ( n = 5). ( e , f ) Astrocytes were transfected with control or the indicated siRNAs (i.e., si-BVR-A or si-HO-1) and subjected to 0.25 mg/mL KRGE for 24 h. HO-1 ( n = 6) or BVR-A ( n = 4) protein levels in cell lysates were detected via Western blotting. * p < 0.05; ** p < 0.01. BVR-A, biliverdin reductase A; GFAP, glial fibrillary acidic protein; HO-1, heme oxygenase 1; KRGE, Korean red ginseng extract; siRNA, small interfering RNA.

Article Snippet: The primary antibodies used in this study were as follows: Tom20 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA; 1:5000, Abcam), Tom22 (1:1000, Santa Cruz Biotechnology), Cyt c (1:3000, BD Pharmingen, San Jose, CA, USA), SIRT1 (1:1000, Santa Cruz Biotechnology), SIRT2 (1:5000, Abcam), SIRT3 (1:1000, Santa Cruz Biotechnology), SIRT4 (1:3000, Thermo Fisher Scientific), SIRT5 (1:1000, Santa Cruz Biotechnology), ERRα (1:3000, Novus Biologicals, Littleton, CO, USA), p-LKB1 (Ser 428 ) (1:2000, Cell Signaling Technology, Danvers, MA, USA), LKB1 (1:2000, Cell Signaling Technology), CaMKKβ (1:1000, Santa Cruz Biotechnology), AMPKα (1:2000, Cell Signaling Technology), p-AMPKα (Thr 172 ) (1:2000, Cell Signaling Technology), SOD2 (1:1000, Santa Cruz Biotechnology), mitochondria (1:10,000, Abcam), HO-1 (1:2000, BD Biosciences, Franklin Lakes, NJ, USA), BVR-A (1:1000, Santa Cruz Biotechnology; 1:3000, Enzo Life Sciences, Farmingdale, NY, USA), and anti-β-actin (1:8000, Sigma-Aldrich).

Techniques: Expressing, Staining, Western Blot, Incubation, Transfection, Small Interfering RNA