Structured Review

Abcam cygb
Overview of the potential mechanisms involved in the DpC-mediated effects on neuroblastoma. DpC increases TNFα expression in neuroblastoma cells, which may (1) activate cytotoxic T cells to destroy tumor cells and/or (2) acts on the TNFα receptor ( TNFR ) to activate down-stream signaling pathways. These include the MAPK/p38/JNK and NF-ĸB signaling cascades, which lead to nuclear transcription of numerous genes, including those that induce apoptosis, as well as cytokines such as TNFα. Activation of TNFR also promotes cleavage of caspase 8, leading to caspase 3 cleavage and subsequent apoptosis. Moreover, DpC is also highly redox active, resulting in the production of reactive oxygen species (ROS) [ 13 ]. The generation of ROS triggers the release of cytochrome c from mitochondria [ 8 ], leading to cleavage of caspase 9, which then also cleaves caspase 3, leading to apoptosis. Further, the increased ROS also leads to upregulation of neuroglobin ( <t>Ngb</t> ) and cytoglobin ( <t>Cygb</t> ) expression as both of these proteins respond to oxidative stress. Together, these molecular effects, which promote apoptosis, could contribute to the anti-cancer activity of DpC in neuroblastoma
Cygb, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews
Price from $9.99 to $1999.99
cygb - by Bioz Stars, 2020-08
90/100 stars

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1) Product Images from "The novel thiosemicarbazone, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), inhibits neuroblastoma growth in vitro and in vivo via multiple mechanisms"

Article Title: The novel thiosemicarbazone, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), inhibits neuroblastoma growth in vitro and in vivo via multiple mechanisms

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-016-0330-x

Overview of the potential mechanisms involved in the DpC-mediated effects on neuroblastoma. DpC increases TNFα expression in neuroblastoma cells, which may (1) activate cytotoxic T cells to destroy tumor cells and/or (2) acts on the TNFα receptor ( TNFR ) to activate down-stream signaling pathways. These include the MAPK/p38/JNK and NF-ĸB signaling cascades, which lead to nuclear transcription of numerous genes, including those that induce apoptosis, as well as cytokines such as TNFα. Activation of TNFR also promotes cleavage of caspase 8, leading to caspase 3 cleavage and subsequent apoptosis. Moreover, DpC is also highly redox active, resulting in the production of reactive oxygen species (ROS) [ 13 ]. The generation of ROS triggers the release of cytochrome c from mitochondria [ 8 ], leading to cleavage of caspase 9, which then also cleaves caspase 3, leading to apoptosis. Further, the increased ROS also leads to upregulation of neuroglobin ( Ngb ) and cytoglobin ( Cygb ) expression as both of these proteins respond to oxidative stress. Together, these molecular effects, which promote apoptosis, could contribute to the anti-cancer activity of DpC in neuroblastoma
Figure Legend Snippet: Overview of the potential mechanisms involved in the DpC-mediated effects on neuroblastoma. DpC increases TNFα expression in neuroblastoma cells, which may (1) activate cytotoxic T cells to destroy tumor cells and/or (2) acts on the TNFα receptor ( TNFR ) to activate down-stream signaling pathways. These include the MAPK/p38/JNK and NF-ĸB signaling cascades, which lead to nuclear transcription of numerous genes, including those that induce apoptosis, as well as cytokines such as TNFα. Activation of TNFR also promotes cleavage of caspase 8, leading to caspase 3 cleavage and subsequent apoptosis. Moreover, DpC is also highly redox active, resulting in the production of reactive oxygen species (ROS) [ 13 ]. The generation of ROS triggers the release of cytochrome c from mitochondria [ 8 ], leading to cleavage of caspase 9, which then also cleaves caspase 3, leading to apoptosis. Further, the increased ROS also leads to upregulation of neuroglobin ( Ngb ) and cytoglobin ( Cygb ) expression as both of these proteins respond to oxidative stress. Together, these molecular effects, which promote apoptosis, could contribute to the anti-cancer activity of DpC in neuroblastoma

Techniques Used: Expressing, Activation Assay, Activity Assay

a Incubation of DpC (25 μM) with SK-N-LP neuroblastoma cells and HK2 non-tumorigenic, immortalized kidney cells induces Cygb and Ngb expression after a 24-h incubation. In these studies, non-tumorigenic, immortalized cell lines (i.e., MSC, H9c2, or HK2), or neoplastic, neuroblastoma (SK-N-LP) cells, were incubated for either 0, 12 or 24 h/37 °C with either control medium ( Con ; no agent added), Dp44mT (25 μM), or DpC (25 μM) and then flow cytometric analysis performed using Flow Jo 8.8.2. Black , red , and blue lines represent the control, or the cells treated with the iron chelators for 12 and 24 h, respectively. Results shown are typical experiments of three performed. b Western blot analysis of Cygb and Ngb expression in SK-N-LP cells following incubation with control media ( Con ), Dp44mT (25 μM), or DpC (25 μM) for 24 h/37 °C. The bands presented in the blots are representative of three repeats and the lanes have been cropped from raw data images containing all three repeats (raw data shown in Additional File 1 ) for clarity (lanes separated by dotted lines ). Densitometry data in ( b ) are presented as the mean ± SEM ( n = 3). * p
Figure Legend Snippet: a Incubation of DpC (25 μM) with SK-N-LP neuroblastoma cells and HK2 non-tumorigenic, immortalized kidney cells induces Cygb and Ngb expression after a 24-h incubation. In these studies, non-tumorigenic, immortalized cell lines (i.e., MSC, H9c2, or HK2), or neoplastic, neuroblastoma (SK-N-LP) cells, were incubated for either 0, 12 or 24 h/37 °C with either control medium ( Con ; no agent added), Dp44mT (25 μM), or DpC (25 μM) and then flow cytometric analysis performed using Flow Jo 8.8.2. Black , red , and blue lines represent the control, or the cells treated with the iron chelators for 12 and 24 h, respectively. Results shown are typical experiments of three performed. b Western blot analysis of Cygb and Ngb expression in SK-N-LP cells following incubation with control media ( Con ), Dp44mT (25 μM), or DpC (25 μM) for 24 h/37 °C. The bands presented in the blots are representative of three repeats and the lanes have been cropped from raw data images containing all three repeats (raw data shown in Additional File 1 ) for clarity (lanes separated by dotted lines ). Densitometry data in ( b ) are presented as the mean ± SEM ( n = 3). * p

Techniques Used: Incubation, Expressing, Flow Cytometry, Western Blot

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    Abcam cygb
    Overview of the potential mechanisms involved in the DpC-mediated effects on neuroblastoma. DpC increases TNFα expression in neuroblastoma cells, which may (1) activate cytotoxic T cells to destroy tumor cells and/or (2) acts on the TNFα receptor ( TNFR ) to activate down-stream signaling pathways. These include the MAPK/p38/JNK and NF-ĸB signaling cascades, which lead to nuclear transcription of numerous genes, including those that induce apoptosis, as well as cytokines such as TNFα. Activation of TNFR also promotes cleavage of caspase 8, leading to caspase 3 cleavage and subsequent apoptosis. Moreover, DpC is also highly redox active, resulting in the production of reactive oxygen species (ROS) [ 13 ]. The generation of ROS triggers the release of cytochrome c from mitochondria [ 8 ], leading to cleavage of caspase 9, which then also cleaves caspase 3, leading to apoptosis. Further, the increased ROS also leads to upregulation of neuroglobin ( <t>Ngb</t> ) and cytoglobin ( <t>Cygb</t> ) expression as both of these proteins respond to oxidative stress. Together, these molecular effects, which promote apoptosis, could contribute to the anti-cancer activity of DpC in neuroblastoma
    Cygb, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cygb/product/Abcam
    Average 90 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    cygb - by Bioz Stars, 2020-08
    90/100 stars
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    Overview of the potential mechanisms involved in the DpC-mediated effects on neuroblastoma. DpC increases TNFα expression in neuroblastoma cells, which may (1) activate cytotoxic T cells to destroy tumor cells and/or (2) acts on the TNFα receptor ( TNFR ) to activate down-stream signaling pathways. These include the MAPK/p38/JNK and NF-ĸB signaling cascades, which lead to nuclear transcription of numerous genes, including those that induce apoptosis, as well as cytokines such as TNFα. Activation of TNFR also promotes cleavage of caspase 8, leading to caspase 3 cleavage and subsequent apoptosis. Moreover, DpC is also highly redox active, resulting in the production of reactive oxygen species (ROS) [ 13 ]. The generation of ROS triggers the release of cytochrome c from mitochondria [ 8 ], leading to cleavage of caspase 9, which then also cleaves caspase 3, leading to apoptosis. Further, the increased ROS also leads to upregulation of neuroglobin ( Ngb ) and cytoglobin ( Cygb ) expression as both of these proteins respond to oxidative stress. Together, these molecular effects, which promote apoptosis, could contribute to the anti-cancer activity of DpC in neuroblastoma

    Journal: Journal of Hematology & Oncology

    Article Title: The novel thiosemicarbazone, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), inhibits neuroblastoma growth in vitro and in vivo via multiple mechanisms

    doi: 10.1186/s13045-016-0330-x

    Figure Lengend Snippet: Overview of the potential mechanisms involved in the DpC-mediated effects on neuroblastoma. DpC increases TNFα expression in neuroblastoma cells, which may (1) activate cytotoxic T cells to destroy tumor cells and/or (2) acts on the TNFα receptor ( TNFR ) to activate down-stream signaling pathways. These include the MAPK/p38/JNK and NF-ĸB signaling cascades, which lead to nuclear transcription of numerous genes, including those that induce apoptosis, as well as cytokines such as TNFα. Activation of TNFR also promotes cleavage of caspase 8, leading to caspase 3 cleavage and subsequent apoptosis. Moreover, DpC is also highly redox active, resulting in the production of reactive oxygen species (ROS) [ 13 ]. The generation of ROS triggers the release of cytochrome c from mitochondria [ 8 ], leading to cleavage of caspase 9, which then also cleaves caspase 3, leading to apoptosis. Further, the increased ROS also leads to upregulation of neuroglobin ( Ngb ) and cytoglobin ( Cygb ) expression as both of these proteins respond to oxidative stress. Together, these molecular effects, which promote apoptosis, could contribute to the anti-cancer activity of DpC in neuroblastoma

    Article Snippet: Antibodies against Ngb and Cygb (Abcam, Cambridge, UK) were kindly provided by Dr. Tan-Un (School of Professional and Continuing Education, The University of Hong Kong, Hong Kong, People’s Republic of China).

    Techniques: Expressing, Activation Assay, Activity Assay

    a Incubation of DpC (25 μM) with SK-N-LP neuroblastoma cells and HK2 non-tumorigenic, immortalized kidney cells induces Cygb and Ngb expression after a 24-h incubation. In these studies, non-tumorigenic, immortalized cell lines (i.e., MSC, H9c2, or HK2), or neoplastic, neuroblastoma (SK-N-LP) cells, were incubated for either 0, 12 or 24 h/37 °C with either control medium ( Con ; no agent added), Dp44mT (25 μM), or DpC (25 μM) and then flow cytometric analysis performed using Flow Jo 8.8.2. Black , red , and blue lines represent the control, or the cells treated with the iron chelators for 12 and 24 h, respectively. Results shown are typical experiments of three performed. b Western blot analysis of Cygb and Ngb expression in SK-N-LP cells following incubation with control media ( Con ), Dp44mT (25 μM), or DpC (25 μM) for 24 h/37 °C. The bands presented in the blots are representative of three repeats and the lanes have been cropped from raw data images containing all three repeats (raw data shown in Additional File 1 ) for clarity (lanes separated by dotted lines ). Densitometry data in ( b ) are presented as the mean ± SEM ( n = 3). * p

    Journal: Journal of Hematology & Oncology

    Article Title: The novel thiosemicarbazone, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), inhibits neuroblastoma growth in vitro and in vivo via multiple mechanisms

    doi: 10.1186/s13045-016-0330-x

    Figure Lengend Snippet: a Incubation of DpC (25 μM) with SK-N-LP neuroblastoma cells and HK2 non-tumorigenic, immortalized kidney cells induces Cygb and Ngb expression after a 24-h incubation. In these studies, non-tumorigenic, immortalized cell lines (i.e., MSC, H9c2, or HK2), or neoplastic, neuroblastoma (SK-N-LP) cells, were incubated for either 0, 12 or 24 h/37 °C with either control medium ( Con ; no agent added), Dp44mT (25 μM), or DpC (25 μM) and then flow cytometric analysis performed using Flow Jo 8.8.2. Black , red , and blue lines represent the control, or the cells treated with the iron chelators for 12 and 24 h, respectively. Results shown are typical experiments of three performed. b Western blot analysis of Cygb and Ngb expression in SK-N-LP cells following incubation with control media ( Con ), Dp44mT (25 μM), or DpC (25 μM) for 24 h/37 °C. The bands presented in the blots are representative of three repeats and the lanes have been cropped from raw data images containing all three repeats (raw data shown in Additional File 1 ) for clarity (lanes separated by dotted lines ). Densitometry data in ( b ) are presented as the mean ± SEM ( n = 3). * p

    Article Snippet: Antibodies against Ngb and Cygb (Abcam, Cambridge, UK) were kindly provided by Dr. Tan-Un (School of Professional and Continuing Education, The University of Hong Kong, Hong Kong, People’s Republic of China).

    Techniques: Incubation, Expressing, Flow Cytometry, Western Blot

    Inhibition of liver fibrosis development in long-term TAA-treated Cygb-TG mice. WT and Cygb-TG (TG) mice were subjected to TAA treatment for 10 weeks (TAA-10w). ( a ) Representative liver images of H E, Sirius Red and Fast Green (SiR-FG) staining for collagen deposition and IF staining for the detection of αSMA (red) and CYGB (green); inset, 6x from the original pictures. P, portal vein; C, central vein. ( b ) Percentages of positive SiR-FG staining area per total areas of whole lobe liver sections were quantified (n = 5). ( c ) Hydroxyproline content in the liver tissue (µg/mg total protein) (n = 9 to 10). ( d ) Percentages of CYGB- and αSMA- positive areas per total areas of whole- lobe liver sections were quantified. ( e ) Immunoblot analysis shows COL1α1, αSMA, CYGB, and mCherry protein expression in the liver tissues of WT and TG mice in images and according to densitometric intensity quantification. GAPDH was used as a loading control and for normalization. Full-length Western blots of one gel are presented in Supplementary Fig. S8 . ( f ) mRNA expression levels of Cygb , mCherry , Col1α1 , αSma , Tgf-β1 Tgf-β3 , and Ppar-γ in the liver were determined by RT-qPCR ( n = 9 to 10). Levels were normalized to Gapdh . ( g ) Liver tissues from WT and TG mice were examined by immunoblotting for phosphorylated- and total SMAD3. The densitometric intensity of phosphorylated-SMAD3 was quantified. GAPDH was used as a loading control and for normalization. Full-length Western blots of one gel are presented in Supplementary Fig. S9 . Data are expressed as the mean ± SD (n = 5). Values are given as the mean ± SD of all experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Journal: Scientific Reports

    Article Title: Selective overexpression of cytoglobin in stellate cells attenuates thioacetamide-induced liver fibrosis in mice

    doi: 10.1038/s41598-018-36215-4

    Figure Lengend Snippet: Inhibition of liver fibrosis development in long-term TAA-treated Cygb-TG mice. WT and Cygb-TG (TG) mice were subjected to TAA treatment for 10 weeks (TAA-10w). ( a ) Representative liver images of H E, Sirius Red and Fast Green (SiR-FG) staining for collagen deposition and IF staining for the detection of αSMA (red) and CYGB (green); inset, 6x from the original pictures. P, portal vein; C, central vein. ( b ) Percentages of positive SiR-FG staining area per total areas of whole lobe liver sections were quantified (n = 5). ( c ) Hydroxyproline content in the liver tissue (µg/mg total protein) (n = 9 to 10). ( d ) Percentages of CYGB- and αSMA- positive areas per total areas of whole- lobe liver sections were quantified. ( e ) Immunoblot analysis shows COL1α1, αSMA, CYGB, and mCherry protein expression in the liver tissues of WT and TG mice in images and according to densitometric intensity quantification. GAPDH was used as a loading control and for normalization. Full-length Western blots of one gel are presented in Supplementary Fig. S8 . ( f ) mRNA expression levels of Cygb , mCherry , Col1α1 , αSma , Tgf-β1 Tgf-β3 , and Ppar-γ in the liver were determined by RT-qPCR ( n = 9 to 10). Levels were normalized to Gapdh . ( g ) Liver tissues from WT and TG mice were examined by immunoblotting for phosphorylated- and total SMAD3. The densitometric intensity of phosphorylated-SMAD3 was quantified. GAPDH was used as a loading control and for normalization. Full-length Western blots of one gel are presented in Supplementary Fig. S9 . Data are expressed as the mean ± SD (n = 5). Values are given as the mean ± SD of all experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Article Snippet: After the membranes were blocked by 5% skim milk, they were probed with the following primary antibodies: anti-CYGB (1:2000; our laboratory), anti-mCherry (1:1000; Abcam, Japan), anti-αSMA (1:2000; DAKO, UK), anti-phosphorylated- and total-SMAD3 (1:1000; Abcam, Japan), anti-phosphorylated- and total-NF-κB (1:1000; Cell Signaling, Japan), or anti-GAPDH (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Inhibition, Mouse Assay, Staining, Expressing, Western Blot, Quantitative RT-PCR

    Overexpression of Cygb attenuates HSC activation. ( a ) Morphology of primary hepatic stellate cells from WT (HSCs Cygb- WT ) and TG mice (HSCs Cygb-TG ) at days 1, 4 and 7. ( b ) Image of lipid droplets and fluorescent mCherry protein in primary HSCs Cygb-TG at day 1. Inset, 3x from original pictures. ( c ) Immunoblot analysis of CYGB, mCherry and αSMA protein expression in primary HSCs Cygb- WT and HSCs Cygb-TG under normal conditions. Right panels show the quantification of the densitometric intensity. Levels were normalized to GAPDH. Full-length Western blots in one gel are presented in Supplementary Fig. S7 . ( d ) mRNA expression levels of Cygb , αSma , Col1α1 and Tgf-β3 in primary HSCs Cygb- WT (white bars) and HSCs Cygb-TG (red bars) at days 1 and 4 were determined by RT-qPCR (n = 5 to 10). Levels were normalized to Gapdh . Values are given as the mean ± SD of all experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Journal: Scientific Reports

    Article Title: Selective overexpression of cytoglobin in stellate cells attenuates thioacetamide-induced liver fibrosis in mice

    doi: 10.1038/s41598-018-36215-4

    Figure Lengend Snippet: Overexpression of Cygb attenuates HSC activation. ( a ) Morphology of primary hepatic stellate cells from WT (HSCs Cygb- WT ) and TG mice (HSCs Cygb-TG ) at days 1, 4 and 7. ( b ) Image of lipid droplets and fluorescent mCherry protein in primary HSCs Cygb-TG at day 1. Inset, 3x from original pictures. ( c ) Immunoblot analysis of CYGB, mCherry and αSMA protein expression in primary HSCs Cygb- WT and HSCs Cygb-TG under normal conditions. Right panels show the quantification of the densitometric intensity. Levels were normalized to GAPDH. Full-length Western blots in one gel are presented in Supplementary Fig. S7 . ( d ) mRNA expression levels of Cygb , αSma , Col1α1 and Tgf-β3 in primary HSCs Cygb- WT (white bars) and HSCs Cygb-TG (red bars) at days 1 and 4 were determined by RT-qPCR (n = 5 to 10). Levels were normalized to Gapdh . Values are given as the mean ± SD of all experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Article Snippet: After the membranes were blocked by 5% skim milk, they were probed with the following primary antibodies: anti-CYGB (1:2000; our laboratory), anti-mCherry (1:1000; Abcam, Japan), anti-αSMA (1:2000; DAKO, UK), anti-phosphorylated- and total-SMAD3 (1:1000; Abcam, Japan), anti-phosphorylated- and total-NF-κB (1:1000; Cell Signaling, Japan), or anti-GAPDH (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Over Expression, Activation Assay, Mouse Assay, Expressing, Western Blot, Quantitative RT-PCR

    Generation of hepatic stellate cell-specific Cygb-transgenic mice. ( a ) DNA construct map for Cygb-mCherry. The Cygb-2A-mCherry reporter was generated with the BAC clone RP23-330N7A, and a partial genomic map of the Cygb gene with coding exons (black boxes), noncoding regions including the promoter of the Cygb gene (light grey boxes), and flanking introns (solid lines) is shown. The 2A-mCherry reporter gene, which was flanked by 110 bp of the upstream sequence of the Cygb gene stop codon and 87 bp of the downstream sequence of its stop codon, was precisely transferred to the Cygb gene. ( b ) Genotyping of Cygb-transgenic ( TG) mice in the F1 generation by Southern blots showed TG mice (lanes 9, 11, 14, and 15) bearing 10 copies of the Cygb transgenes. NC, DNA negative control; PC, DNA positive controls with 1, 3, 10, and 30 copies. ( c ) Genotyping of offspring by real-time qRT-PCR. DNA isolated from tail biopsies of Cygb 10 copies-transgenic founder (TgFD10c) mice was used as a positive control, and DNA from the 1-copy founder (TgFD1c) was used as a reference sample. Relative quantification of Cygb (green bars) and mCherry (red bars) DNA is shown. Mouse numbers BAC 687, 690–694, and 696 were clarified as Cygb-TG, and the remaining mice were WT. The relative number of DNA copies was normalized to Gapdh levels. ( d ) Macroscopic view of multiple organs of WT and Cygb-TG mice under a contrast photo (right panel) and fluorescence images of Cherry (left panel). ( e ) Real-time qRT-PCR analysis shows the Cygb expression levels in multiple organs of WT (white bars) and TG mice (green bars). Transcriptional levels of only mCherry were examined in TG mice (red bars). Gapdh was used as an endogenous control. ( f ) Immunoblot analysis showed the CYGB and mCherry protein levels in multiple organs of WT and TG mice. GAPDH was used as the loading control and for normalization. Full-length Western blots in one gel are presented in Supplementary Fig. S6 . ( g ) Representative imaging of CYGB (green) and mCherry (red) immunofluorescence staining from WT and TG livers. Br, Brain; Li, Liver; Panc, Pancreas; He, Heart; Lg, Lung; Int, Intestine; Sp, Spleen; Kid, Kidney. P, portal vein; C, central vein.

    Journal: Scientific Reports

    Article Title: Selective overexpression of cytoglobin in stellate cells attenuates thioacetamide-induced liver fibrosis in mice

    doi: 10.1038/s41598-018-36215-4

    Figure Lengend Snippet: Generation of hepatic stellate cell-specific Cygb-transgenic mice. ( a ) DNA construct map for Cygb-mCherry. The Cygb-2A-mCherry reporter was generated with the BAC clone RP23-330N7A, and a partial genomic map of the Cygb gene with coding exons (black boxes), noncoding regions including the promoter of the Cygb gene (light grey boxes), and flanking introns (solid lines) is shown. The 2A-mCherry reporter gene, which was flanked by 110 bp of the upstream sequence of the Cygb gene stop codon and 87 bp of the downstream sequence of its stop codon, was precisely transferred to the Cygb gene. ( b ) Genotyping of Cygb-transgenic ( TG) mice in the F1 generation by Southern blots showed TG mice (lanes 9, 11, 14, and 15) bearing 10 copies of the Cygb transgenes. NC, DNA negative control; PC, DNA positive controls with 1, 3, 10, and 30 copies. ( c ) Genotyping of offspring by real-time qRT-PCR. DNA isolated from tail biopsies of Cygb 10 copies-transgenic founder (TgFD10c) mice was used as a positive control, and DNA from the 1-copy founder (TgFD1c) was used as a reference sample. Relative quantification of Cygb (green bars) and mCherry (red bars) DNA is shown. Mouse numbers BAC 687, 690–694, and 696 were clarified as Cygb-TG, and the remaining mice were WT. The relative number of DNA copies was normalized to Gapdh levels. ( d ) Macroscopic view of multiple organs of WT and Cygb-TG mice under a contrast photo (right panel) and fluorescence images of Cherry (left panel). ( e ) Real-time qRT-PCR analysis shows the Cygb expression levels in multiple organs of WT (white bars) and TG mice (green bars). Transcriptional levels of only mCherry were examined in TG mice (red bars). Gapdh was used as an endogenous control. ( f ) Immunoblot analysis showed the CYGB and mCherry protein levels in multiple organs of WT and TG mice. GAPDH was used as the loading control and for normalization. Full-length Western blots in one gel are presented in Supplementary Fig. S6 . ( g ) Representative imaging of CYGB (green) and mCherry (red) immunofluorescence staining from WT and TG livers. Br, Brain; Li, Liver; Panc, Pancreas; He, Heart; Lg, Lung; Int, Intestine; Sp, Spleen; Kid, Kidney. P, portal vein; C, central vein.

    Article Snippet: After the membranes were blocked by 5% skim milk, they were probed with the following primary antibodies: anti-CYGB (1:2000; our laboratory), anti-mCherry (1:1000; Abcam, Japan), anti-αSMA (1:2000; DAKO, UK), anti-phosphorylated- and total-SMAD3 (1:1000; Abcam, Japan), anti-phosphorylated- and total-NF-κB (1:1000; Cell Signaling, Japan), or anti-GAPDH (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Transgenic Assay, Mouse Assay, Construct, Generated, BAC Assay, Sequencing, Negative Control, Quantitative RT-PCR, Isolation, Positive Control, Fluorescence, Expressing, Western Blot, Imaging, Immunofluorescence, Staining

    Inhibition of liver fibrosis development in long-term TAA-treated Cygb-TG mice. WT and Cygb-TG (TG) mice were subjected to TAA treatment for 10 weeks (TAA-10w). ( a ) Representative liver images of H E, Sirius Red and Fast Green (SiR-FG) staining for collagen deposition and IF staining for the detection of αSMA (red) and CYGB (green); inset, 6x from the original pictures. P, portal vein; C, central vein. ( b ) Percentages of positive SiR-FG staining area per total areas of whole lobe liver sections were quantified (n = 5). ( c ) Hydroxyproline content in the liver tissue (µg/mg total protein) (n = 9 to 10). ( d ) Percentages of CYGB- and αSMA- positive areas per total areas of whole- lobe liver sections were quantified. ( e . ( f ) mRNA expression levels of Cygb , mCherry , Col1α1 , αSma , Tgf-β1 Tgf-β3 , and Ppar-γ in the liver were determined by RT-qPCR ( n = 9 to 10). Levels were normalized to Gapdh . ( g . Data are expressed as the mean ± SD (n = 5). Values are given as the mean ± SD of all experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Journal: Scientific Reports

    Article Title: Selective overexpression of cytoglobin in stellate cells attenuates thioacetamide-induced liver fibrosis in mice

    doi: 10.1038/s41598-018-36215-4

    Figure Lengend Snippet: Inhibition of liver fibrosis development in long-term TAA-treated Cygb-TG mice. WT and Cygb-TG (TG) mice were subjected to TAA treatment for 10 weeks (TAA-10w). ( a ) Representative liver images of H E, Sirius Red and Fast Green (SiR-FG) staining for collagen deposition and IF staining for the detection of αSMA (red) and CYGB (green); inset, 6x from the original pictures. P, portal vein; C, central vein. ( b ) Percentages of positive SiR-FG staining area per total areas of whole lobe liver sections were quantified (n = 5). ( c ) Hydroxyproline content in the liver tissue (µg/mg total protein) (n = 9 to 10). ( d ) Percentages of CYGB- and αSMA- positive areas per total areas of whole- lobe liver sections were quantified. ( e . ( f ) mRNA expression levels of Cygb , mCherry , Col1α1 , αSma , Tgf-β1 Tgf-β3 , and Ppar-γ in the liver were determined by RT-qPCR ( n = 9 to 10). Levels were normalized to Gapdh . ( g . Data are expressed as the mean ± SD (n = 5). Values are given as the mean ± SD of all experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Article Snippet: After the membranes were blocked by 5% skim milk, they were probed with the following primary antibodies: anti-CYGB (1:2000; our laboratory), anti-mCherry (1:1000; Abcam, Japan), anti-αSMA (1:2000; DAKO, UK), anti-phosphorylated- and total-SMAD3 (1:1000; Abcam, Japan), anti-phosphorylated- and total-NF-κB (1:1000; Cell Signaling, Japan), or anti-GAPDH (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Inhibition, Mouse Assay, Staining, Expressing, Quantitative RT-PCR

    Overexpression of Cygb attenuates HSC activation. ( a ) Morphology of primary hepatic stellate cells from WT (HSCs Cygb- WT ) and TG mice (HSCs Cygb-TG ) at days 1, 4 and 7. ( b ) Image of lipid droplets and fluorescent mCherry protein in primary HSCs Cygb-TG at day 1. Inset, 3x from original pictures. ( c ) Immunoblot analysis of CYGB, mCherry and αSMA protein expression in primary HSCs Cygb- WT and HSCs Cygb-TG . ( d ) mRNA expression levels of Cygb , αSma , Col1α1 and Tgf-β3 in primary HSCs Cygb- WT (white bars) and HSCs Cygb-TG (red bars) at days 1 and 4 were determined by RT-qPCR (n = 5 to 10). Levels were normalized to Gapdh . Values are given as the mean ± SD of all experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Journal: Scientific Reports

    Article Title: Selective overexpression of cytoglobin in stellate cells attenuates thioacetamide-induced liver fibrosis in mice

    doi: 10.1038/s41598-018-36215-4

    Figure Lengend Snippet: Overexpression of Cygb attenuates HSC activation. ( a ) Morphology of primary hepatic stellate cells from WT (HSCs Cygb- WT ) and TG mice (HSCs Cygb-TG ) at days 1, 4 and 7. ( b ) Image of lipid droplets and fluorescent mCherry protein in primary HSCs Cygb-TG at day 1. Inset, 3x from original pictures. ( c ) Immunoblot analysis of CYGB, mCherry and αSMA protein expression in primary HSCs Cygb- WT and HSCs Cygb-TG . ( d ) mRNA expression levels of Cygb , αSma , Col1α1 and Tgf-β3 in primary HSCs Cygb- WT (white bars) and HSCs Cygb-TG (red bars) at days 1 and 4 were determined by RT-qPCR (n = 5 to 10). Levels were normalized to Gapdh . Values are given as the mean ± SD of all experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Article Snippet: After the membranes were blocked by 5% skim milk, they were probed with the following primary antibodies: anti-CYGB (1:2000; our laboratory), anti-mCherry (1:1000; Abcam, Japan), anti-αSMA (1:2000; DAKO, UK), anti-phosphorylated- and total-SMAD3 (1:1000; Abcam, Japan), anti-phosphorylated- and total-NF-κB (1:1000; Cell Signaling, Japan), or anti-GAPDH (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Over Expression, Activation Assay, Mouse Assay, Expressing, Quantitative RT-PCR

    Generation of hepatic stellate cell-specific Cygb-transgenic mice. ( a ) DNA construct map for Cygb-mCherry. The Cygb-2A-mCherry reporter was generated with the BAC clone RP23-330N7A, and a partial genomic map of the Cygb gene with coding exons (black boxes), noncoding regions including the promoter of the Cygb gene (light grey boxes), and flanking introns (solid lines) is shown. The 2A-mCherry reporter gene, which was flanked by 110 bp of the upstream sequence of the Cygb gene stop codon and 87 bp of the downstream sequence of its stop codon, was precisely transferred to the Cygb gene. ( b ) Genotyping of Cygb-transgenic ( TG) mice in the F1 generation by Southern blots showed TG mice (lanes 9, 11, 14, and 15) bearing 10 copies of the Cygb transgenes. NC, DNA negative control; PC, DNA positive controls with 1, 3, 10, and 30 copies. ( c ) Genotyping of offspring by real-time qRT-PCR. DNA isolated from tail biopsies of Cygb 10 copies-transgenic founder (TgFD10c) mice was used as a positive control, and DNA from the 1-copy founder (TgFD1c) was used as a reference sample. Relative quantification of Cygb (green bars) and mCherry (red bars) DNA is shown. Mouse numbers BAC 687, 690–694, and 696 were clarified as Cygb-TG, and the remaining mice were WT. The relative number of DNA copies was normalized to Gapdh levels. ( d ) Macroscopic view of multiple organs of WT and Cygb-TG mice under a contrast photo (right panel) and fluorescence images of Cherry (left panel). ( e ) Real-time qRT-PCR analysis shows the Cygb expression levels in multiple organs of WT (white bars) and TG mice (green bars). Transcriptional levels of only mCherry were examined in TG mice (red bars). Gapdh was used as an endogenous control. ( f . ( g ) Representative imaging of CYGB (green) and mCherry (red) immunofluorescence staining from WT and TG livers. Br, Brain; Li, Liver; Panc, Pancreas; He, Heart; Lg, Lung; Int, Intestine; Sp, Spleen; Kid, Kidney. P, portal vein; C, central vein.

    Journal: Scientific Reports

    Article Title: Selective overexpression of cytoglobin in stellate cells attenuates thioacetamide-induced liver fibrosis in mice

    doi: 10.1038/s41598-018-36215-4

    Figure Lengend Snippet: Generation of hepatic stellate cell-specific Cygb-transgenic mice. ( a ) DNA construct map for Cygb-mCherry. The Cygb-2A-mCherry reporter was generated with the BAC clone RP23-330N7A, and a partial genomic map of the Cygb gene with coding exons (black boxes), noncoding regions including the promoter of the Cygb gene (light grey boxes), and flanking introns (solid lines) is shown. The 2A-mCherry reporter gene, which was flanked by 110 bp of the upstream sequence of the Cygb gene stop codon and 87 bp of the downstream sequence of its stop codon, was precisely transferred to the Cygb gene. ( b ) Genotyping of Cygb-transgenic ( TG) mice in the F1 generation by Southern blots showed TG mice (lanes 9, 11, 14, and 15) bearing 10 copies of the Cygb transgenes. NC, DNA negative control; PC, DNA positive controls with 1, 3, 10, and 30 copies. ( c ) Genotyping of offspring by real-time qRT-PCR. DNA isolated from tail biopsies of Cygb 10 copies-transgenic founder (TgFD10c) mice was used as a positive control, and DNA from the 1-copy founder (TgFD1c) was used as a reference sample. Relative quantification of Cygb (green bars) and mCherry (red bars) DNA is shown. Mouse numbers BAC 687, 690–694, and 696 were clarified as Cygb-TG, and the remaining mice were WT. The relative number of DNA copies was normalized to Gapdh levels. ( d ) Macroscopic view of multiple organs of WT and Cygb-TG mice under a contrast photo (right panel) and fluorescence images of Cherry (left panel). ( e ) Real-time qRT-PCR analysis shows the Cygb expression levels in multiple organs of WT (white bars) and TG mice (green bars). Transcriptional levels of only mCherry were examined in TG mice (red bars). Gapdh was used as an endogenous control. ( f . ( g ) Representative imaging of CYGB (green) and mCherry (red) immunofluorescence staining from WT and TG livers. Br, Brain; Li, Liver; Panc, Pancreas; He, Heart; Lg, Lung; Int, Intestine; Sp, Spleen; Kid, Kidney. P, portal vein; C, central vein.

    Article Snippet: After the membranes were blocked by 5% skim milk, they were probed with the following primary antibodies: anti-CYGB (1:2000; our laboratory), anti-mCherry (1:1000; Abcam, Japan), anti-αSMA (1:2000; DAKO, UK), anti-phosphorylated- and total-SMAD3 (1:1000; Abcam, Japan), anti-phosphorylated- and total-NF-κB (1:1000; Cell Signaling, Japan), or anti-GAPDH (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Transgenic Assay, Mouse Assay, Construct, Generated, BAC Assay, Sequencing, Negative Control, Quantitative RT-PCR, Isolation, Positive Control, Fluorescence, Expressing, Imaging, Immunofluorescence, Staining

    Cygb-immunofluorescence in a frontal section of the mouse brain. (A) Habenular nuclei and anterior hippocampal formation at the level of approximately interaural +2.1 mm, corresponding to Figure 45 of the mouse brain atlas. Strong staining of neurons in the medial habenular nuclei (MHb), while those in the lateral nuclei (LHb) were less stained. (B) Higher magnification from the medial CA1 region of the hippocampus: strong immunostaining of neurons in the pyramidal cell layer (Py). A few neurons from the oriens layer (Or) and stratum radiatum (Rad) were also stained. (C) Habenular nuclei and fasciculus retroflexus (fr) at the level of approximately interaural +1.7 mm, corresponding to Figure 48 of the mouse brain atlas, (D) Higher magnification demonstrating fibers entering the fasciculus retroflexus. (E) ventral aspect of the lateral geniculate nucleus (VLG) between the optic tract (opt) and the medial lemniscus (ml). (F) Scattered neurons in the posterior hypothalamic area (PH) and the dorsomedial hypothalamic nucleus (DM). Abbreviations: 3V, third ventricle; CA1, Cornu ammonis 1; cp, cerebral peduncle; DG, dentate gyrus; DLG, dorsal lateral geniculate nucleus; LPMR, lateral posterior thalamic nucleus, mediorostral part; mt, mamillothalamic tract.

    Journal: Frontiers in Neuroanatomy

    Article Title: Distribution of Cytoglobin in the Mouse Brain

    doi: 10.3389/fnana.2016.00047

    Figure Lengend Snippet: Cygb-immunofluorescence in a frontal section of the mouse brain. (A) Habenular nuclei and anterior hippocampal formation at the level of approximately interaural +2.1 mm, corresponding to Figure 45 of the mouse brain atlas. Strong staining of neurons in the medial habenular nuclei (MHb), while those in the lateral nuclei (LHb) were less stained. (B) Higher magnification from the medial CA1 region of the hippocampus: strong immunostaining of neurons in the pyramidal cell layer (Py). A few neurons from the oriens layer (Or) and stratum radiatum (Rad) were also stained. (C) Habenular nuclei and fasciculus retroflexus (fr) at the level of approximately interaural +1.7 mm, corresponding to Figure 48 of the mouse brain atlas, (D) Higher magnification demonstrating fibers entering the fasciculus retroflexus. (E) ventral aspect of the lateral geniculate nucleus (VLG) between the optic tract (opt) and the medial lemniscus (ml). (F) Scattered neurons in the posterior hypothalamic area (PH) and the dorsomedial hypothalamic nucleus (DM). Abbreviations: 3V, third ventricle; CA1, Cornu ammonis 1; cp, cerebral peduncle; DG, dentate gyrus; DLG, dorsal lateral geniculate nucleus; LPMR, lateral posterior thalamic nucleus, mediorostral part; mt, mamillothalamic tract.

    Article Snippet: For double-immunostaining experiments, selected sections were incubated in primary Cygb-antibody as described above and simultaneously, in either a polyclonal sheep antibody raised against (nNOS, 1:50, Abcam, Cambridge, UK) or a mouse monoclonal antibody raised against tyrosine hydroxylase (TH, 1:200, Chemicon, Temecula, CA, USA).

    Techniques: Immunofluorescence, Staining, Immunostaining