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  • 99
    Thermo Fisher gene exp hmbs hs00609297 m1
    Expression profiling of ELOVLs in human and mouse lung SCC A . mRNA levels of ELOVL1-7 were analyzed by RT-qPCR in 30 human SCC tissues and 9 pools (14-15 different samples) of matched normal tissues. A normalization factor corresponding to the geometric mean of Ct values of ESD, <t>HMBS,</t> POLR2A, PSMB4, TBP and UBC was used for subsequent ΔCt calculation. B . Tumor versus normal lung tissue (n=3) from the L-IKKα KA/KA SCC mouse model were collected and mRNA levels of ELOVL1-7 were measured using RT-qPCR. 18S was used for normalization. ELOVL2 and 3 were not detected. Data represent mean (log2) ± standard error. * p
    Gene Exp Hmbs Hs00609297 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 210 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore hmb pp
    Vγ9 − Vδ2 + T cells are clonally and phenotypically distinct from Vγ9 + Vδ2 + T cells. a Frequency of Vγ9 chain usage in Vδ2 + TCR repertoire sequencing data from adult peripheral blood ( n = 7). b Identification of Vδ2 + T cells in CD3 + T cells (left) and Vγ9 − cells within Vδ2 + T cells (right) from cord blood ( n = 5). c Frequency of Vγ9 − and Vγ9 + cells in Vδ2 + T cells from cord blood (left; n = 5) and adult peripheral blood (right; n = 18). d Frequency of Vγ9 − Vδ2 + T cells ( n = 18) and NKT cells (αGalcer/CD1d + ; n = 5) in adult peripheral blood. e Vγ9 − Vδ2 + T cells identified by TCR Vδ2 antibody clones (B6 and 123R3) in matched adult peripheral blood donors ( n = 5). f Proliferation by <t>CFSE</t> dilution of CD8 + αβ and γδ T-cell populations from PBMC treated with medium alone, 10 nM <t>HMB-PP</t> or 5 μg/ml anti-CD3/CD28 ( n = 5). g CD27 and CD45RA T-cell memory marker expression profiles on Vγ9 − Vδ2 + and Vδ1 + T cells from adult peripheral blood (top row: Vδ2, n = 14; Vδ1, n = 14) and cord blood (bottom row: Vδ2 and Vδ1, n = 5). h Vδ2 + TCR and CD27 expression levels, i naive and j effector T-cell marker expression on donor matched γδ T-cell populations from adult peripheral blood ( n = 3). k Sorted CD3 + T cells were incubated for 72 h with cytokines or anti-CD3/CD28 beads. Vγ9 − Vδ2 + T cells were then assessed for the upregulation of CD25 ( n = 3). l Comparison of CDR3γ sequence sharing in γδ T-cell repertoires from adult peripheral blood (Vδ1, n = 20; Vδ2, n = 7) and cord blood (Vδ1, n = 5; Vδ2, n = 3). m CDR3γ sequence analysis of single cell sorted Vγ9 + and Vγ9 − Vδ2 + T cells, public sequences are coloured (black, shared sequences from deep sequencing); graph shows Vγ usage of Vγ9 − Vδ2 + TCR sequences from each donor. Error bars indicate means ± SEM; ** P
    Hmb Pp, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies hmb 45
    Representative histological images of H E staining and immunohistochemistry for protein expression (stained in brown) in each group. All immunohistological parameters showed significantly higher values in the more severe group. a , c , e , g , i , and k : the less severe group; b , d , f , h , j , and l : the more severe group. A and B: H E staining showing the distribution of cysts and LAM-cells nodules (arrows) in each group (magnification: 5×). c and d : SMA expression in LAM-cells (magnification: C, 7× and D, 7×). e and f : <t>HMB-45</t> expression in LAM-cells (magnification: E, 20× and F, 40×). g and h : VEGF-D expression in LAM-cells (magnification: G, 100× and H, 100×). i and j : MMP-9 expression in LAM-cells (magnification: I, 25× and J, 25×). k and l : mTOR expression in LAM-cells (magnification: K, 35× and L, 35×)
    Hmb 45, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 283 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    R&D Systems recombinant human hmgb1
    Down-regulation of clusterin expression after inhibition of NFκB-p65 phosphorylation. ( A ) Recombinant <t>HMGB1,</t> added to DU145 tumor cells, enhanced NFκB-p65 phosphorylation within 1–5 min, as analyzed by western blot. ( B,C ) DU145 tumor cells, pretreated with medium, 20 μM PS1145 or 10 μM BAY11-7082 for 1 h, were cultured with HMGB1 for another 24 h. Cells were then lysed for analysis of clusterin mRNA expression by Q-PCR or clusterin protein expression by western blot. Analysis of phosphorylated NFκB-p65 was included to check for effectiveness of the inhibitors.
    Recombinant Human Hmgb1, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 194 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Ventana Medical hmb 45
    <t>HMB-45</t> staining RPE cells and scattered melanocytes in the uninvolved choroid (original magnification X400).
    Hmb 45, supplied by Ventana Medical, used in various techniques. Bioz Stars score: 91/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher gene exp hmbs mm01143545 m1
    Effect of reference genes on the calculated expression of Irf4 and Zbtb20 .The relative expression of Irf4 ( a ) and Zbtb20 ( b ) in aLN and iLN were normalized with Actb , Ywhaz , Gapdh , Tbp , Ubc , Hprt or <t>Hmbs</t> by RT-qPCR after 1 and 7 days of the 1st and 3rd vaccination (V1D1, V1D7, V3D1 and V3D7, respectively). Data are presented as mean + SEM. Comparison of ΔΔCq values by one-way ANOVA and Tukey’s multiple comparison test was performed. Experimental groups identified with different symbols were significantly different from each other ( P
    Gene Exp Hmbs Mm01143545 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher gene exp hmbs hs00609296 g1
    Decrease of furin activity delays tumor growth of Rh30 xenografts in vivo A. Schematic overview of Rh30 xenograft model. 3-4 NOD/Scid mice per group were fed with or without DOX-supplemented food starting 7 days prior engraftment. Mice were then engrafted s.c. with 2.5 million Rh30 cells. Tumor growth was monitored over time and mice were sacrificed once a tumor reached a size of 1000 mm 3 . B. Absence of furin activity delays initial tumor growth. Tumor growth rate was monitored by caliper measurements. Data represent mean tumor size of 3-4 mice per group. C. Furin expression is reduced upon induction of furin specific shRNA. Tumor tissue was collected upon sacrifice of mice, total RNA isolated and furin mRNA levels analyzed by qRT-PCR. Expression levels relative to <t>HMBS</t> are shown.
    Gene Exp Hmbs Hs00609296 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Zhongshan Golden Bridge Company hmb 45
    <t>HMB-45</t> expression in normal Langerhans cells without skin damage caused by melanoma (magnification, ×400).
    Hmb 45, supplied by Zhongshan Golden Bridge Company, used in various techniques. Bioz Stars score: 91/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher gene exp hmbs mm00660262 g1
    Absence of GDNF modulation by estradiol replacement in ovariectomized female mice. (A) Mouse experimental model. Ovariectomized (ovx) animals were separated in four groups and received either sham implant (O) or E 2 implant (Oi, E1 and E5) at surgery time. Three weeks later, mice received Vehicle (O and Oi), a single s.c. E 2 injection (E1), or five E 2 injections over 5 days (E5). Animals were sacrificed 24 hours after s.c. injection (O, Oi, E1) or 4–5 hours after the last injection (E5). (B) Vertical bar graph showing the mean ± SEM of plasma E 2 in O, Oi, E1 and E5 mice. This shows that the estrogen replacement protocol induced an increase in circulating E 2 in ovx mice. (C) Western blot analysis of phosphorylated Akt (pAkt) showing a representative blot (left), and pAkt / Akt ratio is shown with a vertical bar graph (right). The greatest difference is found between O and E1 (p = 0.053). (D) Gdnf mRNA expression, reported as Gdnf1-2 relative to bActin , in the striatum of wild-type ( Gdnf +/+ ) and heterozygous mice ( Gdnf +/- ). (E) GDNF protein content expressed as pg / mg total protein in Gdnf +/+ and Gdnf +/- striatum. (F) Scatter plot diagram showing the positive correlation (ρ = 0.72, p = 0.002) between Gdnf mRNA (shown in D) and GDNF protein levels (shown in E) in Gdnf +/+ and Gdnf +/- striata. This data shows that GDNF protein concentration measured by ELISA is consistent with Gdnf gene expression level. (G) QPCR analysis of Gdnf expression measured by Gdnf1-2 relative to Parv mRNA (left graph) or <t>Hmbs</t> mRNA (right). (H) ELISA analysis of GDNF protein levels showing no difference between the experimental groups. All data are presented as the mean ± SEM. *, p
    Gene Exp Hmbs Mm00660262 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Thermo Fisher gene exp hmbs rn00565886 m1
    mRNA quantification in amphetamine and saline treated animals. Normalized quantities for D 1 , D 2 and PDE10A acquired in amphetamine and saline treated. GUSB and <t>HMBS</t> were used for the normalization of quantities values. Data are presented as mean ± SD. No significant difference in mRNA levels of amphetamine and saline treated animals could be found
    Gene Exp Hmbs Rn00565886 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Novocastra hmb 45
    Immunohistochemical stains showing cytoplasmic positivity to <t>HMB-45</t> (2A ×400) and melan A (2B ×400).
    Hmb 45, supplied by Novocastra, used in various techniques. Bioz Stars score: 91/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Agilent technologies anti hmb 45
    Immunohistochemical stains showing cytoplasmic positivity to <t>HMB-45</t> (2A ×400) and melan A (2B ×400).
    Anti Hmb 45, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 88/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abcam hmb 45
    Immunohistochemical stains showing cytoplasmic positivity to <t>HMB-45</t> (2A ×400) and melan A (2B ×400).
    Hmb 45, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    NOEX Spolka hmb supplementation
    Immunohistochemical stains showing cytoplasmic positivity to <t>HMB-45</t> (2A ×400) and melan A (2B ×400).
    Hmb Supplementation, supplied by NOEX Spolka, used in various techniques. Bioz Stars score: 94/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abbott Laboratories hmb
    Immunohistochemical stains showing cytoplasmic positivity to <t>HMB-45</t> (2A ×400) and melan A (2B ×400).
    Hmb, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher hydroxymethylbilane synthase hmbs
    Immunohistochemical stains showing cytoplasmic positivity to <t>HMB-45</t> (2A ×400) and melan A (2B ×400).
    Hydroxymethylbilane Synthase Hmbs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Novocastra mouse monoclonal antibody against hmb 45
    Immunohistochemical stains showing cytoplasmic positivity to <t>HMB-45</t> (2A ×400) and melan A (2B ×400).
    Mouse Monoclonal Antibody Against Hmb 45, supplied by Novocastra, used in various techniques. Bioz Stars score: 85/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Enzo Biochem hmb 45
    Immunohistochemical stains showing cytoplasmic positivity to <t>HMB-45</t> (2A ×400) and melan A (2B ×400).
    Hmb 45, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hmb  (COMSOL)
    90
    COMSOL hmb
    Linearity of radial, axial, and current stiffness of <t>HMB</t> from <t>COMSOL</t> (gray color) and MBTR (black color) on Geometry 6, the optimized HMB. Radial stiffness: −5.1 (MBTR) and −5.9 (FEA) N/mm, Axial stiffness 3.1 (MBTR) and 3.2 (FEA) N/mm,
    Hmb, supplied by COMSOL, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hmb  (Nissen)
    91
    Nissen hmb
    Linearity of radial, axial, and current stiffness of <t>HMB</t> from <t>COMSOL</t> (gray color) and MBTR (black color) on Geometry 6, the optimized HMB. Radial stiffness: −5.1 (MBTR) and −5.9 (FEA) N/mm, Axial stiffness 3.1 (MBTR) and 3.2 (FEA) N/mm,
    Hmb, supplied by Nissen, used in various techniques. Bioz Stars score: 91/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam anti hmbs antibody epr8105
    Linearity of radial, axial, and current stiffness of <t>HMB</t> from <t>COMSOL</t> (gray color) and MBTR (black color) on Geometry 6, the optimized HMB. Radial stiffness: −5.1 (MBTR) and −5.9 (FEA) N/mm, Axial stiffness 3.1 (MBTR) and 3.2 (FEA) N/mm,
    Anti Hmbs Antibody Epr8105, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher gene exp hmbs hs00609293 g1
    Screening of CYP2W1 inducers in the colon adenocarcinoma cell line HCC2998. CYP2W1 mRNA levels following the 48 h treatment with several selected drugs. The CYP2W1 transcript expression was normalized by housekeeping genes EIF2B2, <t>HMBS</t> and PPIA and related to control (vehicle alone, either DMSO or ethanol depending on the drug). Data are presented as mean±SEM of at least two experiments in at least two replicates per experiment. * p
    Gene Exp Hmbs Hs00609293 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Agilent technologies human melanoma black hmb 45
    Immunohistochemical staining showing (a) Positivity of tumor cells with S-100 and negativity for (b) Pan cytokeratin, (c) HMB 45, and (d) Vimentin (×400)
    Human Melanoma Black Hmb 45, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression profiling of ELOVLs in human and mouse lung SCC A . mRNA levels of ELOVL1-7 were analyzed by RT-qPCR in 30 human SCC tissues and 9 pools (14-15 different samples) of matched normal tissues. A normalization factor corresponding to the geometric mean of Ct values of ESD, HMBS, POLR2A, PSMB4, TBP and UBC was used for subsequent ΔCt calculation. B . Tumor versus normal lung tissue (n=3) from the L-IKKα KA/KA SCC mouse model were collected and mRNA levels of ELOVL1-7 were measured using RT-qPCR. 18S was used for normalization. ELOVL2 and 3 were not detected. Data represent mean (log2) ± standard error. * p

    Journal: Oncotarget

    Article Title: Phospholipid profiling identifies acyl chain elongation as a ubiquitous trait and potential target for the treatment of lung squamous cell carcinoma

    doi: 10.18632/oncotarget.7179

    Figure Lengend Snippet: Expression profiling of ELOVLs in human and mouse lung SCC A . mRNA levels of ELOVL1-7 were analyzed by RT-qPCR in 30 human SCC tissues and 9 pools (14-15 different samples) of matched normal tissues. A normalization factor corresponding to the geometric mean of Ct values of ESD, HMBS, POLR2A, PSMB4, TBP and UBC was used for subsequent ΔCt calculation. B . Tumor versus normal lung tissue (n=3) from the L-IKKα KA/KA SCC mouse model were collected and mRNA levels of ELOVL1-7 were measured using RT-qPCR. 18S was used for normalization. ELOVL2 and 3 were not detected. Data represent mean (log2) ± standard error. * p

    Article Snippet: Commercially available primers (TaqMan Gene Expression Assay; Life Technologies) for ELOVL1-7 (Hs00249277_m1, Hs00214936_m1, Hs00537016_m1, Hs00224122_m1, Hs01094704_m1, Hs00907564_m1, Hs00405150_m1), and the housekeeping genes ESD (Hs00382667_m1), HMBS (Hs00609297_m1), POLR2A (Hs00172187_m1), PSMB4 (Hs00160598_m1), TBP (Hs00427621_m1) and UBC (Hs00824723_m1) were used. qPCR was conducted on an OpenArray instrument (Applied Biosystems, Carlsbad, California, USA). qPCR was carried out on 30 SCC tissues and 9 pools (14-15 different samples) of matched normal tissues.

    Techniques: Expressing, Quantitative RT-PCR

    Vγ9 − Vδ2 + T cells are clonally and phenotypically distinct from Vγ9 + Vδ2 + T cells. a Frequency of Vγ9 chain usage in Vδ2 + TCR repertoire sequencing data from adult peripheral blood ( n = 7). b Identification of Vδ2 + T cells in CD3 + T cells (left) and Vγ9 − cells within Vδ2 + T cells (right) from cord blood ( n = 5). c Frequency of Vγ9 − and Vγ9 + cells in Vδ2 + T cells from cord blood (left; n = 5) and adult peripheral blood (right; n = 18). d Frequency of Vγ9 − Vδ2 + T cells ( n = 18) and NKT cells (αGalcer/CD1d + ; n = 5) in adult peripheral blood. e Vγ9 − Vδ2 + T cells identified by TCR Vδ2 antibody clones (B6 and 123R3) in matched adult peripheral blood donors ( n = 5). f Proliferation by CFSE dilution of CD8 + αβ and γδ T-cell populations from PBMC treated with medium alone, 10 nM HMB-PP or 5 μg/ml anti-CD3/CD28 ( n = 5). g CD27 and CD45RA T-cell memory marker expression profiles on Vγ9 − Vδ2 + and Vδ1 + T cells from adult peripheral blood (top row: Vδ2, n = 14; Vδ1, n = 14) and cord blood (bottom row: Vδ2 and Vδ1, n = 5). h Vδ2 + TCR and CD27 expression levels, i naive and j effector T-cell marker expression on donor matched γδ T-cell populations from adult peripheral blood ( n = 3). k Sorted CD3 + T cells were incubated for 72 h with cytokines or anti-CD3/CD28 beads. Vγ9 − Vδ2 + T cells were then assessed for the upregulation of CD25 ( n = 3). l Comparison of CDR3γ sequence sharing in γδ T-cell repertoires from adult peripheral blood (Vδ1, n = 20; Vδ2, n = 7) and cord blood (Vδ1, n = 5; Vδ2, n = 3). m CDR3γ sequence analysis of single cell sorted Vγ9 + and Vγ9 − Vδ2 + T cells, public sequences are coloured (black, shared sequences from deep sequencing); graph shows Vγ usage of Vγ9 − Vδ2 + TCR sequences from each donor. Error bars indicate means ± SEM; ** P

    Journal: Nature Communications

    Article Title: The human Vδ2+ T-cell compartment comprises distinct innate-like Vγ9+ and adaptive Vγ9- subsets

    doi: 10.1038/s41467-018-04076-0

    Figure Lengend Snippet: Vγ9 − Vδ2 + T cells are clonally and phenotypically distinct from Vγ9 + Vδ2 + T cells. a Frequency of Vγ9 chain usage in Vδ2 + TCR repertoire sequencing data from adult peripheral blood ( n = 7). b Identification of Vδ2 + T cells in CD3 + T cells (left) and Vγ9 − cells within Vδ2 + T cells (right) from cord blood ( n = 5). c Frequency of Vγ9 − and Vγ9 + cells in Vδ2 + T cells from cord blood (left; n = 5) and adult peripheral blood (right; n = 18). d Frequency of Vγ9 − Vδ2 + T cells ( n = 18) and NKT cells (αGalcer/CD1d + ; n = 5) in adult peripheral blood. e Vγ9 − Vδ2 + T cells identified by TCR Vδ2 antibody clones (B6 and 123R3) in matched adult peripheral blood donors ( n = 5). f Proliferation by CFSE dilution of CD8 + αβ and γδ T-cell populations from PBMC treated with medium alone, 10 nM HMB-PP or 5 μg/ml anti-CD3/CD28 ( n = 5). g CD27 and CD45RA T-cell memory marker expression profiles on Vγ9 − Vδ2 + and Vδ1 + T cells from adult peripheral blood (top row: Vδ2, n = 14; Vδ1, n = 14) and cord blood (bottom row: Vδ2 and Vδ1, n = 5). h Vδ2 + TCR and CD27 expression levels, i naive and j effector T-cell marker expression on donor matched γδ T-cell populations from adult peripheral blood ( n = 3). k Sorted CD3 + T cells were incubated for 72 h with cytokines or anti-CD3/CD28 beads. Vγ9 − Vδ2 + T cells were then assessed for the upregulation of CD25 ( n = 3). l Comparison of CDR3γ sequence sharing in γδ T-cell repertoires from adult peripheral blood (Vδ1, n = 20; Vδ2, n = 7) and cord blood (Vδ1, n = 5; Vδ2, n = 3). m CDR3γ sequence analysis of single cell sorted Vγ9 + and Vγ9 − Vδ2 + T cells, public sequences are coloured (black, shared sequences from deep sequencing); graph shows Vγ usage of Vγ9 − Vδ2 + TCR sequences from each donor. Error bars indicate means ± SEM; ** P

    Article Snippet: For proliferation of T cells, PBMC were labelled with 0.3 µM carboxyfluorescein succinimidyl ester (CFSE) (eBioscience) and cultured with 10 nM HMB-PP (Sigma) or CD3/CD28 T activator beads (Invitrogen) for 7 days in RPMI-1640 medium (Invitrogen) supplemented with 2 mM L-glutamine, 1% sodium pyruvate, 50 μg/ml penicillin/streptomycin (Invitrogen) and 10% fetal calf serum (Sigma).

    Techniques: Sequencing, Clone Assay, Marker, Expressing, Incubation

    Representative histological images of H E staining and immunohistochemistry for protein expression (stained in brown) in each group. All immunohistological parameters showed significantly higher values in the more severe group. a , c , e , g , i , and k : the less severe group; b , d , f , h , j , and l : the more severe group. A and B: H E staining showing the distribution of cysts and LAM-cells nodules (arrows) in each group (magnification: 5×). c and d : SMA expression in LAM-cells (magnification: C, 7× and D, 7×). e and f : HMB-45 expression in LAM-cells (magnification: E, 20× and F, 40×). g and h : VEGF-D expression in LAM-cells (magnification: G, 100× and H, 100×). i and j : MMP-9 expression in LAM-cells (magnification: I, 25× and J, 25×). k and l : mTOR expression in LAM-cells (magnification: K, 35× and L, 35×)

    Journal: Respiratory Research

    Article Title: Immunohistological features related to functional impairment in lymphangioleiomyomatosis

    doi: 10.1186/s12931-018-0797-9

    Figure Lengend Snippet: Representative histological images of H E staining and immunohistochemistry for protein expression (stained in brown) in each group. All immunohistological parameters showed significantly higher values in the more severe group. a , c , e , g , i , and k : the less severe group; b , d , f , h , j , and l : the more severe group. A and B: H E staining showing the distribution of cysts and LAM-cells nodules (arrows) in each group (magnification: 5×). c and d : SMA expression in LAM-cells (magnification: C, 7× and D, 7×). e and f : HMB-45 expression in LAM-cells (magnification: E, 20× and F, 40×). g and h : VEGF-D expression in LAM-cells (magnification: G, 100× and H, 100×). i and j : MMP-9 expression in LAM-cells (magnification: I, 25× and J, 25×). k and l : mTOR expression in LAM-cells (magnification: K, 35× and L, 35×)

    Article Snippet: Sections were incubated with the primary antibody overnight at 4 °C using the following antibodies: SMA, clone 1A4, 1:1000, Dako, California, United States; HMB-45, clone HMB-45, 1:400, Dako, California, United States; VEGF-D, clone 78,923, 1:7000, R & D Systems, United Kingdom.

    Techniques: Staining, Immunohistochemistry, Expressing, Laser Capture Microdissection

    Down-regulation of clusterin expression after inhibition of NFκB-p65 phosphorylation. ( A ) Recombinant HMGB1, added to DU145 tumor cells, enhanced NFκB-p65 phosphorylation within 1–5 min, as analyzed by western blot. ( B,C ) DU145 tumor cells, pretreated with medium, 20 μM PS1145 or 10 μM BAY11-7082 for 1 h, were cultured with HMGB1 for another 24 h. Cells were then lysed for analysis of clusterin mRNA expression by Q-PCR or clusterin protein expression by western blot. Analysis of phosphorylated NFκB-p65 was included to check for effectiveness of the inhibitors.

    Journal: Scientific Reports

    Article Title: HMGB1 induction of clusterin creates a chemoresistant niche in human prostate tumor cells

    doi: 10.1038/srep15085

    Figure Lengend Snippet: Down-regulation of clusterin expression after inhibition of NFκB-p65 phosphorylation. ( A ) Recombinant HMGB1, added to DU145 tumor cells, enhanced NFκB-p65 phosphorylation within 1–5 min, as analyzed by western blot. ( B,C ) DU145 tumor cells, pretreated with medium, 20 μM PS1145 or 10 μM BAY11-7082 for 1 h, were cultured with HMGB1 for another 24 h. Cells were then lysed for analysis of clusterin mRNA expression by Q-PCR or clusterin protein expression by western blot. Analysis of phosphorylated NFκB-p65 was included to check for effectiveness of the inhibitors.

    Article Snippet: Induction of clusterin DU145 tumor cells, plated to confluency overnight in 6 well plates, were treated with 1.0 μg/ml recombinant human HMGB1 (R & D systems) or supernatants harvested from DTX-treated DU145 tumor cells.

    Techniques: Expressing, Inhibition, Recombinant, Western Blot, Cell Culture, Polymerase Chain Reaction

    HMGB1 induces clusterin via TLR4 and RAGE. ( A ) DU145 tumor cells, pretreated with DTX for 6 h, were washed and then reincubated in fresh medium for another 24 h. Supernatants were collected and added to freshly-plated DU145 tumor cells for 24 h, either untreated or pretreated with anti-HMGB1 for 30 min. ( B ) Flow cytometric analysis of DU145 tumor cells treated with DTX for 24 h demonstrated no change for RAGE and TLR4 expression. ( C ) DU145 tumor cells, untreated or pretreated with anti-TLR4, anti-RAGE or both antibodies for 30 min, were exposed to recombinant HMGB1 or supernatants from DTX-pretreated tumor cells. After 24 h, the cells were lysed and analyzed for presence for clusterin by western blot.

    Journal: Scientific Reports

    Article Title: HMGB1 induction of clusterin creates a chemoresistant niche in human prostate tumor cells

    doi: 10.1038/srep15085

    Figure Lengend Snippet: HMGB1 induces clusterin via TLR4 and RAGE. ( A ) DU145 tumor cells, pretreated with DTX for 6 h, were washed and then reincubated in fresh medium for another 24 h. Supernatants were collected and added to freshly-plated DU145 tumor cells for 24 h, either untreated or pretreated with anti-HMGB1 for 30 min. ( B ) Flow cytometric analysis of DU145 tumor cells treated with DTX for 24 h demonstrated no change for RAGE and TLR4 expression. ( C ) DU145 tumor cells, untreated or pretreated with anti-TLR4, anti-RAGE or both antibodies for 30 min, were exposed to recombinant HMGB1 or supernatants from DTX-pretreated tumor cells. After 24 h, the cells were lysed and analyzed for presence for clusterin by western blot.

    Article Snippet: Induction of clusterin DU145 tumor cells, plated to confluency overnight in 6 well plates, were treated with 1.0 μg/ml recombinant human HMGB1 (R & D systems) or supernatants harvested from DTX-treated DU145 tumor cells.

    Techniques: Flow Cytometry, Expressing, Recombinant, Western Blot

    Dying tumor cells release HMGB1 which induces clusterin from neighboring live tumor cells. DU145 tumor cells were untreated or treated with DTX for 24 h and then dually stained for HMGB1 (green) and clusterin (red), as well as DAPI (blue) to visualize the nucleus. Live tumor cells (indicated by white arrows) in medium culture have intact round blue nucleus ( A ), intranuclear green speckled HMGB1 ( B ) and little cytoplasmic red clusterin ( C ). This separation of HMGB1 and clusterin is cleary seen in the merged picture ( D ). However, DTX treatment causes a change in the dynamics of the tumor cells. A DTX-treated dying tumor cell, indicated by a red arrow in the center of a cell cluster, has an irregular blue nuclei ( E ), and has released its green HMGB1 from the nucleus into the cytoplasm and surrounding medium ( F ). The dying cell is surrounded by 4 live tumor cells (white arrows), which still have intact nuclei with intranuclear speckled green HMGB1. More remarkably, these 4 live tumor cells now express high levels of cytoplasmic red clusterin. Thus, the HMGB1 released from the central dying tumor cells appear to have induced clusterin from the 4 neighboring tumor cells.

    Journal: Scientific Reports

    Article Title: HMGB1 induction of clusterin creates a chemoresistant niche in human prostate tumor cells

    doi: 10.1038/srep15085

    Figure Lengend Snippet: Dying tumor cells release HMGB1 which induces clusterin from neighboring live tumor cells. DU145 tumor cells were untreated or treated with DTX for 24 h and then dually stained for HMGB1 (green) and clusterin (red), as well as DAPI (blue) to visualize the nucleus. Live tumor cells (indicated by white arrows) in medium culture have intact round blue nucleus ( A ), intranuclear green speckled HMGB1 ( B ) and little cytoplasmic red clusterin ( C ). This separation of HMGB1 and clusterin is cleary seen in the merged picture ( D ). However, DTX treatment causes a change in the dynamics of the tumor cells. A DTX-treated dying tumor cell, indicated by a red arrow in the center of a cell cluster, has an irregular blue nuclei ( E ), and has released its green HMGB1 from the nucleus into the cytoplasm and surrounding medium ( F ). The dying cell is surrounded by 4 live tumor cells (white arrows), which still have intact nuclei with intranuclear speckled green HMGB1. More remarkably, these 4 live tumor cells now express high levels of cytoplasmic red clusterin. Thus, the HMGB1 released from the central dying tumor cells appear to have induced clusterin from the 4 neighboring tumor cells.

    Article Snippet: Induction of clusterin DU145 tumor cells, plated to confluency overnight in 6 well plates, were treated with 1.0 μg/ml recombinant human HMGB1 (R & D systems) or supernatants harvested from DTX-treated DU145 tumor cells.

    Techniques: Staining

    HMGB1 confers chemoresistance via TLR4 and RAGE. ( A ) DU145 tumor cells, untreated or pretreated with control IgG, anti-TLR4, anti-RAGE, or both antibodies for 30 min, were exposed to recombinant HMGB1 for 24 h. Then, DTX was added for another 48 h prior to staining with methylene blue for visualization of live tumor cells. ( B ) The same experiment was conducted with r ecombinant HMGB1 substituted for supernatants from DTX-treated tumor cells. ( C ) DU145 tumor cells pretreated with recombinant HMGB1 were transfected with antisense-scramble or antisense-clusterin for 24 h, and then cells were cultured in medium with or without DTX for another 48 h prior to staining with methylene blue.

    Journal: Scientific Reports

    Article Title: HMGB1 induction of clusterin creates a chemoresistant niche in human prostate tumor cells

    doi: 10.1038/srep15085

    Figure Lengend Snippet: HMGB1 confers chemoresistance via TLR4 and RAGE. ( A ) DU145 tumor cells, untreated or pretreated with control IgG, anti-TLR4, anti-RAGE, or both antibodies for 30 min, were exposed to recombinant HMGB1 for 24 h. Then, DTX was added for another 48 h prior to staining with methylene blue for visualization of live tumor cells. ( B ) The same experiment was conducted with r ecombinant HMGB1 substituted for supernatants from DTX-treated tumor cells. ( C ) DU145 tumor cells pretreated with recombinant HMGB1 were transfected with antisense-scramble or antisense-clusterin for 24 h, and then cells were cultured in medium with or without DTX for another 48 h prior to staining with methylene blue.

    Article Snippet: Induction of clusterin DU145 tumor cells, plated to confluency overnight in 6 well plates, were treated with 1.0 μg/ml recombinant human HMGB1 (R & D systems) or supernatants harvested from DTX-treated DU145 tumor cells.

    Techniques: Recombinant, Staining, Transfection, Cell Culture

    Clusterin blocks DTX-mediated apoptosis by sequestering Bax from mitochondria. DU145 tumor cells, pretreated with or without recombinant HMGB1 for 24 h, were treated with medium or DTX for 4 h. Mitochondria was visualized with MitoTracker Red, and activated Bax with 6A7 monoclonal antibody (green). Cells were examined by confocal microscopy (magnification, ×630) for colocalization of activated Bax with mitochondria. One representative image of three independent experiments is shown.

    Journal: Scientific Reports

    Article Title: HMGB1 induction of clusterin creates a chemoresistant niche in human prostate tumor cells

    doi: 10.1038/srep15085

    Figure Lengend Snippet: Clusterin blocks DTX-mediated apoptosis by sequestering Bax from mitochondria. DU145 tumor cells, pretreated with or without recombinant HMGB1 for 24 h, were treated with medium or DTX for 4 h. Mitochondria was visualized with MitoTracker Red, and activated Bax with 6A7 monoclonal antibody (green). Cells were examined by confocal microscopy (magnification, ×630) for colocalization of activated Bax with mitochondria. One representative image of three independent experiments is shown.

    Article Snippet: Induction of clusterin DU145 tumor cells, plated to confluency overnight in 6 well plates, were treated with 1.0 μg/ml recombinant human HMGB1 (R & D systems) or supernatants harvested from DTX-treated DU145 tumor cells.

    Techniques: Recombinant, Confocal Microscopy

    HMGB1 induces clusterin from tumor cells and is released by dying tumor cells. ( A ) DU145 prostate tumor cells, incubated with recombinant human HMGB1 (rhHMGB1) for 24–48 h, were lysed and analyzed by western blot for presence of clusterin, and for β-actin for equal loading. ( B ) DU145 tumor cells were treated with docetaxel (DTX) for 1–4 days and their supernatants were evaluated for HMGB1 by ELISA. ( C ) DU145 tumor cells were treated with the indicated chemotherapeutic agents for 24 h and the supernatants analyzed for HMGB1.

    Journal: Scientific Reports

    Article Title: HMGB1 induction of clusterin creates a chemoresistant niche in human prostate tumor cells

    doi: 10.1038/srep15085

    Figure Lengend Snippet: HMGB1 induces clusterin from tumor cells and is released by dying tumor cells. ( A ) DU145 prostate tumor cells, incubated with recombinant human HMGB1 (rhHMGB1) for 24–48 h, were lysed and analyzed by western blot for presence of clusterin, and for β-actin for equal loading. ( B ) DU145 tumor cells were treated with docetaxel (DTX) for 1–4 days and their supernatants were evaluated for HMGB1 by ELISA. ( C ) DU145 tumor cells were treated with the indicated chemotherapeutic agents for 24 h and the supernatants analyzed for HMGB1.

    Article Snippet: Induction of clusterin DU145 tumor cells, plated to confluency overnight in 6 well plates, were treated with 1.0 μg/ml recombinant human HMGB1 (R & D systems) or supernatants harvested from DTX-treated DU145 tumor cells.

    Techniques: Incubation, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay

    Membrane attack complex (MAC) deposition is reduced by high-mobility group box 1 (HMGB1) neutralizing antibody (Ab) in middle cerebral arterial occlusion (MCAO) ischemia mouse model. (A) HMGB1 staining of ICR mouse brain 4 h after MCAO. Contralateral and ipsilateral brain sections were stained for HMGB1 and collagen (Col) IV, which visualizes most blood vessels and capillaries. The images captured at low and high magnifications are shown. (B) Pearson’s coefficient for the overlapping of HMGB1 and DAPI in low magnification of three fields was calculated. Error bars are mean ± SD. ** P

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: Membrane attack complex (MAC) deposition is reduced by high-mobility group box 1 (HMGB1) neutralizing antibody (Ab) in middle cerebral arterial occlusion (MCAO) ischemia mouse model. (A) HMGB1 staining of ICR mouse brain 4 h after MCAO. Contralateral and ipsilateral brain sections were stained for HMGB1 and collagen (Col) IV, which visualizes most blood vessels and capillaries. The images captured at low and high magnifications are shown. (B) Pearson’s coefficient for the overlapping of HMGB1 and DAPI in low magnification of three fields was calculated. Error bars are mean ± SD. ** P

    Article Snippet: The HMGB1- or human IgG-coated microspheres were washed three times with GVB2+ buffer and incubated with 10% NHS for 30 min at 37°C.

    Techniques: Staining

    High-mobility group box 1 (HMGB1)-induced complement activation model. HMGB1 protein is actively secreted or passively released by stress injury on cells or organs in the forms of three different redox statuses, which may differently modulate immunological activities. Extracellular HMGB1 can activate the classical pathway of complement system in an antibody (Ab)-independent manner after binding to C1q, resulting in forming C5b-9 membrane attack complexes (MAC) where HMGB1 is accumulated. Thus, HMGB1-induced complement activation is proposed to be able to exacerbate sterile inflammation.

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-induced complement activation model. HMGB1 protein is actively secreted or passively released by stress injury on cells or organs in the forms of three different redox statuses, which may differently modulate immunological activities. Extracellular HMGB1 can activate the classical pathway of complement system in an antibody (Ab)-independent manner after binding to C1q, resulting in forming C5b-9 membrane attack complexes (MAC) where HMGB1 is accumulated. Thus, HMGB1-induced complement activation is proposed to be able to exacerbate sterile inflammation.

    Article Snippet: The HMGB1- or human IgG-coated microspheres were washed three times with GVB2+ buffer and incubated with 10% NHS for 30 min at 37°C.

    Techniques: Activation Assay, Binding Assay

    High-mobility group box 1 (HMGB1)-mediated cleavage of C4 and C3 and the formation of C5b-9 [membrane attack complex (MAC)]. (A) Measurement of C4b. A microtiter plate was coated with Euk-HMGB1 (10 µg/ml, R D) and incubated with various concentrations of normal human serum (NHS) to test the deposition of C4b using an enzyme-linked immunosorbent assay (ELISA) (left). Human IgG (10 µg/ml) was used as the positive control (right). (B) To determine C3a and iC3b, the cleavage product of C3b. Fifty microliters of NHS (diluted 1:5) was incubated with increasing amounts of HMGB1 for 30 min at 37°C, then performed Western blot analysis using anti-C3/iC3b or anti-C3a antibodies. Aggregated human IgG and Ig-free BSA served as the positive and negative controls, respectively. Ig light chain (IgL) was used as input control. (C,D) Measurements of complement activation products of C3c, the cleaved form of C3b, and C5b-9. Biotinylated HMGB1s were incubated with streptavidin-coated microspheres in 10% NHS, which was pre-absorbed with non-coated microspheres, for 30 min at 37°C. Immunofluorescence staining was performed with anti-HMGB1 and fluorescein isothiocyanate-conjugated anti-C3c antibody (Ab). For C5b-9 deposition, immunofluorescence staining was performed with anti-HMGB1 (green) and anti-C5b-9 Ab (red). Biotinylated human IgG was used as the positive control. (E) A microtiter plate was coated with HMGB1 (10 µg/ml) and incubated with various concentrations of NHS to test the deposition of C5b-9 using an ELISA. Aggregated human IgG was used as the positive control. (F) C1q-dependent MAC formation. A microtiter plate was coated with HMGB1 (10 µg/ml) and incubated with various concentrations of NHS or C1q-depleted human serum (C1q-dep HS) to test the deposition of MAC formation using an ELISA for C5b-9. To restore the effect of C1q, 20 µg/ml of C1q was added to C1q-depleted human serum. ns: not significant. (G,H) Measurement of complement activation after HMGB1 injection in mice. C57BL/6 mice were intravenously injected with 100 µg of HMGB1 or PBS to observe if HMGB1 can activate complement in vivo ( N = 3 per group). Blood samples were collected at 0, 30, and 90 min after the injection. iC3b (arrow) was detected using Western blot analysis of serum sample from one representative mouse. Relative band intensity of iC3b was analyzed. All data shown here are representative of at least three independent experiments with similar results. Error bars are mean ± SD. * P

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-mediated cleavage of C4 and C3 and the formation of C5b-9 [membrane attack complex (MAC)]. (A) Measurement of C4b. A microtiter plate was coated with Euk-HMGB1 (10 µg/ml, R D) and incubated with various concentrations of normal human serum (NHS) to test the deposition of C4b using an enzyme-linked immunosorbent assay (ELISA) (left). Human IgG (10 µg/ml) was used as the positive control (right). (B) To determine C3a and iC3b, the cleavage product of C3b. Fifty microliters of NHS (diluted 1:5) was incubated with increasing amounts of HMGB1 for 30 min at 37°C, then performed Western blot analysis using anti-C3/iC3b or anti-C3a antibodies. Aggregated human IgG and Ig-free BSA served as the positive and negative controls, respectively. Ig light chain (IgL) was used as input control. (C,D) Measurements of complement activation products of C3c, the cleaved form of C3b, and C5b-9. Biotinylated HMGB1s were incubated with streptavidin-coated microspheres in 10% NHS, which was pre-absorbed with non-coated microspheres, for 30 min at 37°C. Immunofluorescence staining was performed with anti-HMGB1 and fluorescein isothiocyanate-conjugated anti-C3c antibody (Ab). For C5b-9 deposition, immunofluorescence staining was performed with anti-HMGB1 (green) and anti-C5b-9 Ab (red). Biotinylated human IgG was used as the positive control. (E) A microtiter plate was coated with HMGB1 (10 µg/ml) and incubated with various concentrations of NHS to test the deposition of C5b-9 using an ELISA. Aggregated human IgG was used as the positive control. (F) C1q-dependent MAC formation. A microtiter plate was coated with HMGB1 (10 µg/ml) and incubated with various concentrations of NHS or C1q-depleted human serum (C1q-dep HS) to test the deposition of MAC formation using an ELISA for C5b-9. To restore the effect of C1q, 20 µg/ml of C1q was added to C1q-depleted human serum. ns: not significant. (G,H) Measurement of complement activation after HMGB1 injection in mice. C57BL/6 mice were intravenously injected with 100 µg of HMGB1 or PBS to observe if HMGB1 can activate complement in vivo ( N = 3 per group). Blood samples were collected at 0, 30, and 90 min after the injection. iC3b (arrow) was detected using Western blot analysis of serum sample from one representative mouse. Relative band intensity of iC3b was analyzed. All data shown here are representative of at least three independent experiments with similar results. Error bars are mean ± SD. * P

    Article Snippet: The HMGB1- or human IgG-coated microspheres were washed three times with GVB2+ buffer and incubated with 10% NHS for 30 min at 37°C.

    Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Positive Control, Western Blot, Activation Assay, Immunofluorescence, Staining, Injection, Mouse Assay, In Vivo

    High-mobility group box 1 (HMGB1) B box activates complement cascade. (A) Schematic overview of recombinant HMGB1 proteins used: wild-type (WT) HMGB1, boxes A (aa 1-79) and B (aa 88-162), ΔC-HMGB1 (aa 1-185), and ΔN-HMGB1 (aa 11-215). (B,C) Complement consumption assessment. WT HMGB1 proteins (B) or HMGB1 variants (C) were incubated with the diluted normal human serum (NHS) containing CH 50 activity in GVB 2+ buffer for 30 min at 37°C. After complement consumption by HMGB1, sRBCs were added in the consumed NHS for 30 min at 37°C. Aggregated human IgG and IgG-free BSA (each 30 µg/ml) were used as the positive and negative controls, respectively. Data shown are the mean ± SD of three independent repeats. * P

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: High-mobility group box 1 (HMGB1) B box activates complement cascade. (A) Schematic overview of recombinant HMGB1 proteins used: wild-type (WT) HMGB1, boxes A (aa 1-79) and B (aa 88-162), ΔC-HMGB1 (aa 1-185), and ΔN-HMGB1 (aa 11-215). (B,C) Complement consumption assessment. WT HMGB1 proteins (B) or HMGB1 variants (C) were incubated with the diluted normal human serum (NHS) containing CH 50 activity in GVB 2+ buffer for 30 min at 37°C. After complement consumption by HMGB1, sRBCs were added in the consumed NHS for 30 min at 37°C. Aggregated human IgG and IgG-free BSA (each 30 µg/ml) were used as the positive and negative controls, respectively. Data shown are the mean ± SD of three independent repeats. * P

    Article Snippet: The HMGB1- or human IgG-coated microspheres were washed three times with GVB2+ buffer and incubated with 10% NHS for 30 min at 37°C.

    Techniques: Recombinant, Incubation, Activity Assay

    High-mobility group box 1 (HMGB1)-mediated membrane attack complex (MAC) formation and its effect to cell signaling. (A,B) MEFs were cultured in DMEM containing 10% normal human serum (NHS) in the presence or absence of 1 µg/ml HMGB1 for 1 h at 37°C. Sublytic MAC proteins were stained using anti-C5b-9 antibody (Ab) (red) and observed by confocal microscopy. Blue: DAPI. Heat-inactivated (HI) NHS was used. Scale bar, 10 µm (A) . MEFs were incubated with 10% NHS in the presence of different concentrations of HMGB1, and then the mean relative intensity of fluorescence of 10 visual fields was calculated (B) . Error bars are mean ± SD. * P

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-mediated membrane attack complex (MAC) formation and its effect to cell signaling. (A,B) MEFs were cultured in DMEM containing 10% normal human serum (NHS) in the presence or absence of 1 µg/ml HMGB1 for 1 h at 37°C. Sublytic MAC proteins were stained using anti-C5b-9 antibody (Ab) (red) and observed by confocal microscopy. Blue: DAPI. Heat-inactivated (HI) NHS was used. Scale bar, 10 µm (A) . MEFs were incubated with 10% NHS in the presence of different concentrations of HMGB1, and then the mean relative intensity of fluorescence of 10 visual fields was calculated (B) . Error bars are mean ± SD. * P

    Article Snippet: The HMGB1- or human IgG-coated microspheres were washed three times with GVB2+ buffer and incubated with 10% NHS for 30 min at 37°C.

    Techniques: Cell Culture, Staining, Confocal Microscopy, Incubation, Fluorescence

    High-mobility group box 1 (HMGB1)-induced complement activation is decreased in C1q-deficient mice of APAP-induced hepatotoxicity model. (A) Overnight fasted wild-type (WT) or C1q −/− mice (9–10 weeks old, male) were intraperitoneally (i.p.) treated with 400 mg/kg of APAP or saline. After 0, 6, and 24 h, mouse serum samples were collected. Serum alanine aminotransferase (ALT) activity (left) and C3 levels (right) were determined using ALT color endpoint assay and enzyme-linked immunosorbent assay, respectively. Each dot represents each mouse with triplicates per sample (four or five mice per group). (B,C) Mice were euthanized 24 h after APAP administration, and the left medial lobe of liver was fixed in 4% paraformaldehyde and 30% sucrose. Liver tissue was stained with hematoxylin and eosin for evaluation of necrosis and hemorrhage (B) . Tissue-Tec OCT-embedded liver was stained with anti-C1q and anti-HMGB1 antibodies (Abs) for immunofluorescence analysis (C) . (D) Serum HMGB1 protein from WT and C1q −/− mice was detected using Western blot analysis 24 h after APAP injection. (E) Anti-HMGB1 (2G7) or mouse IgG (5 μg/mouse) was i.p. administrated immediately after APAP administration to block HMGB1. After 24 h, mouse serum samples were collected and ALT activity (left panel) and concentration of C3 (right panel) were determined. Saline was used as a negative control. (F) HMGB1 was i.p. injected with sRAGE (5 µg/mouse) as described in (E) . N = 4–5 mice/group. Data = mean ± SEM. Student’s t -test (unpaired two-tailed) was used to calculate the P -value. All scale bars, 20 µm. Data are representative of three (A–E) or two (F) independent experiments.

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: High-mobility group box 1 (HMGB1)-induced complement activation is decreased in C1q-deficient mice of APAP-induced hepatotoxicity model. (A) Overnight fasted wild-type (WT) or C1q −/− mice (9–10 weeks old, male) were intraperitoneally (i.p.) treated with 400 mg/kg of APAP or saline. After 0, 6, and 24 h, mouse serum samples were collected. Serum alanine aminotransferase (ALT) activity (left) and C3 levels (right) were determined using ALT color endpoint assay and enzyme-linked immunosorbent assay, respectively. Each dot represents each mouse with triplicates per sample (four or five mice per group). (B,C) Mice were euthanized 24 h after APAP administration, and the left medial lobe of liver was fixed in 4% paraformaldehyde and 30% sucrose. Liver tissue was stained with hematoxylin and eosin for evaluation of necrosis and hemorrhage (B) . Tissue-Tec OCT-embedded liver was stained with anti-C1q and anti-HMGB1 antibodies (Abs) for immunofluorescence analysis (C) . (D) Serum HMGB1 protein from WT and C1q −/− mice was detected using Western blot analysis 24 h after APAP injection. (E) Anti-HMGB1 (2G7) or mouse IgG (5 μg/mouse) was i.p. administrated immediately after APAP administration to block HMGB1. After 24 h, mouse serum samples were collected and ALT activity (left panel) and concentration of C3 (right panel) were determined. Saline was used as a negative control. (F) HMGB1 was i.p. injected with sRAGE (5 µg/mouse) as described in (E) . N = 4–5 mice/group. Data = mean ± SEM. Student’s t -test (unpaired two-tailed) was used to calculate the P -value. All scale bars, 20 µm. Data are representative of three (A–E) or two (F) independent experiments.

    Article Snippet: The HMGB1- or human IgG-coated microspheres were washed three times with GVB2+ buffer and incubated with 10% NHS for 30 min at 37°C.

    Techniques: Activation Assay, Mouse Assay, Activity Assay, End Point Assay, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence, Western Blot, Injection, Blocking Assay, Concentration Assay, Negative Control, Two Tailed Test

    Binding of high-mobility group box 1 (HMGB1) to C1q. (A) Purified C1q protein (10 µg/ml) was immobilized on a microtiter plate, and different concentrations of HMGB1 protein were added for an enzyme-linked immunosorbent assay (ELISA). Buffer was used as a negative control. Data are representative of three independent experiments. Error bars are mean ± SD. * P

    Journal: Frontiers in Immunology

    Article Title: High-Mobility Group Box 1-Induced Complement Activation Causes Sterile Inflammation

    doi: 10.3389/fimmu.2018.00705

    Figure Lengend Snippet: Binding of high-mobility group box 1 (HMGB1) to C1q. (A) Purified C1q protein (10 µg/ml) was immobilized on a microtiter plate, and different concentrations of HMGB1 protein were added for an enzyme-linked immunosorbent assay (ELISA). Buffer was used as a negative control. Data are representative of three independent experiments. Error bars are mean ± SD. * P

    Article Snippet: The HMGB1- or human IgG-coated microspheres were washed three times with GVB2+ buffer and incubated with 10% NHS for 30 min at 37°C.

    Techniques: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Negative Control

    Damaged tissue induces macrophage mtDNA fragmentation. ( a ) Separation of DNA fragments by agarose gel electrophoresis in BCM treated with or without nuclease. ( b ) Confocal images and quantification of TUNEL and Hoechst staining of BMDM treated for 24 h with nuclease pretreated or non-pretreated BCM (40 μ l/ml). ( c ) Immunofluorescence images showing the colocalization and quantification of colocalization of endosome (EEA1; green) and fragmented DNA (TUNEL; red) in BMDM treated with 40 μ l/ml BCM for 24 h. ( d ) Confocal immunofluorescence images showing colocalization and quantification of colocalization of mitochondria (MitoTracker; green) and fragmented DNA (TUNEL; red) in BMDM treated with BCM (40 μ l/ml) for 24 h. ( e ) Quantification of fold induction of mtDNA measured by RT-PCR in BMDM treated with 0–40 μ l/ml BCM or 40 μ l/ml nuclease-pretreated BCM for 6 h. ( f ) mtDNA fold induction and ( g ) mtDNA damage measured by RT-PCR in BMDM treated with BCM (40 μ l/ml) for 0, 6, or 24 h. ( h ) Western blot for CIRP and HMGB1 expression in BCM. ( i ) Confocal images and quantification of TUNEL and Hoechst staining of BMDM challenged with BCM (40 μ l/ml), CIRP (10 μ g/ml), or HMGB1 (10 μ g/ml) for 24 h. ( j ) mtDNA fold induction measured by RT-PCR in BMDM treated with 0–10 μ g/ml CIRP or HMGB1 for 6 h. ( k ) mtDNA damage measured by RT-PCR in BMDM stimulated with BCM (40 μ l/ml), CIRP (10 μ g/ml), or HMGB1 (10 μ g/ml), which combined with or without IgG isotype antibody (10 μ g/ml) or CIRP neutralizing antibody (10 μ g/ml) for 24 h. ( l ) Quantification of mtDNA by RT-PCR with three specific mitochondrial primers (Mt-1/2/3) in J774.2 cells treated with 100 ng/ml ethidium bromide (EtBr) for 0–14 days. ( m ) mtDNA fold induction quantified by RT-PCR in J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, and stimulation with BCM (40 μ l/ml) for 0, 6, and 24 h. ( n ) Confocal images and quantification of fragemented DNA (TUNEL; red) and Hoechst nuclear staining of J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, and stimulated with BCM (40 μ l/ml) for 0, 6, and 24 h. All results are representative of three independent experiments. The graph shows the mean and S.E.M., n =3. Significances between groups were determined by using independent samples two-tailed Student’s t -test. * P

    Journal: Cell Death & Disease

    Article Title: Cold-inducible RNA-binding protein through TLR4 signaling induces mitochondrial DNA fragmentation and regulates macrophage cell death after trauma

    doi: 10.1038/cddis.2017.187

    Figure Lengend Snippet: Damaged tissue induces macrophage mtDNA fragmentation. ( a ) Separation of DNA fragments by agarose gel electrophoresis in BCM treated with or without nuclease. ( b ) Confocal images and quantification of TUNEL and Hoechst staining of BMDM treated for 24 h with nuclease pretreated or non-pretreated BCM (40 μ l/ml). ( c ) Immunofluorescence images showing the colocalization and quantification of colocalization of endosome (EEA1; green) and fragmented DNA (TUNEL; red) in BMDM treated with 40 μ l/ml BCM for 24 h. ( d ) Confocal immunofluorescence images showing colocalization and quantification of colocalization of mitochondria (MitoTracker; green) and fragmented DNA (TUNEL; red) in BMDM treated with BCM (40 μ l/ml) for 24 h. ( e ) Quantification of fold induction of mtDNA measured by RT-PCR in BMDM treated with 0–40 μ l/ml BCM or 40 μ l/ml nuclease-pretreated BCM for 6 h. ( f ) mtDNA fold induction and ( g ) mtDNA damage measured by RT-PCR in BMDM treated with BCM (40 μ l/ml) for 0, 6, or 24 h. ( h ) Western blot for CIRP and HMGB1 expression in BCM. ( i ) Confocal images and quantification of TUNEL and Hoechst staining of BMDM challenged with BCM (40 μ l/ml), CIRP (10 μ g/ml), or HMGB1 (10 μ g/ml) for 24 h. ( j ) mtDNA fold induction measured by RT-PCR in BMDM treated with 0–10 μ g/ml CIRP or HMGB1 for 6 h. ( k ) mtDNA damage measured by RT-PCR in BMDM stimulated with BCM (40 μ l/ml), CIRP (10 μ g/ml), or HMGB1 (10 μ g/ml), which combined with or without IgG isotype antibody (10 μ g/ml) or CIRP neutralizing antibody (10 μ g/ml) for 24 h. ( l ) Quantification of mtDNA by RT-PCR with three specific mitochondrial primers (Mt-1/2/3) in J774.2 cells treated with 100 ng/ml ethidium bromide (EtBr) for 0–14 days. ( m ) mtDNA fold induction quantified by RT-PCR in J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, and stimulation with BCM (40 μ l/ml) for 0, 6, and 24 h. ( n ) Confocal images and quantification of fragemented DNA (TUNEL; red) and Hoechst nuclear staining of J774.2 cells +/− pretreatment with 100 ng/ml EtBr for 7 days, and stimulated with BCM (40 μ l/ml) for 0, 6, and 24 h. All results are representative of three independent experiments. The graph shows the mean and S.E.M., n =3. Significances between groups were determined by using independent samples two-tailed Student’s t -test. * P

    Article Snippet: Recombinant HMGB1 (1690-HMB-050) was from R & D Systems.

    Techniques: Agarose Gel Electrophoresis, TUNEL Assay, Staining, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Two Tailed Test

    HMB-45 staining RPE cells and scattered melanocytes in the uninvolved choroid (original magnification X400).

    Journal: ecancermedicalscience

    Article Title: Diagnostic value of SOX-10 immunohistochemical staining for the detection of uveal melanoma

    doi: 10.3332/ecancer.2015.566

    Figure Lengend Snippet: HMB-45 staining RPE cells and scattered melanocytes in the uninvolved choroid (original magnification X400).

    Article Snippet: For HMB-45, a mouse monoclonal HMB-45 antibody was used (HMB-45: 790-4366, Ventana validated protocol).

    Techniques: Staining

    SOX-10 and HMB-45 expression in uveal melanoma (original magnification X400 [A through D]) (A) Diffuse nuclear SOX-10 positivity (B) Focal SOX-10 staining (C) Diffuse HMB-45 cytoplasmic staining pattern (D) Focal HMB-45 positivity.

    Journal: ecancermedicalscience

    Article Title: Diagnostic value of SOX-10 immunohistochemical staining for the detection of uveal melanoma

    doi: 10.3332/ecancer.2015.566

    Figure Lengend Snippet: SOX-10 and HMB-45 expression in uveal melanoma (original magnification X400 [A through D]) (A) Diffuse nuclear SOX-10 positivity (B) Focal SOX-10 staining (C) Diffuse HMB-45 cytoplasmic staining pattern (D) Focal HMB-45 positivity.

    Article Snippet: For HMB-45, a mouse monoclonal HMB-45 antibody was used (HMB-45: 790-4366, Ventana validated protocol).

    Techniques: Expressing, Staining

    (A) Negative HMB-45 in a case of uveal melanoma (B) SOX-10 showing diffuse strong positivity of the same case. (original magnification X400 A B).

    Journal: ecancermedicalscience

    Article Title: Diagnostic value of SOX-10 immunohistochemical staining for the detection of uveal melanoma

    doi: 10.3332/ecancer.2015.566

    Figure Lengend Snippet: (A) Negative HMB-45 in a case of uveal melanoma (B) SOX-10 showing diffuse strong positivity of the same case. (original magnification X400 A B).

    Article Snippet: For HMB-45, a mouse monoclonal HMB-45 antibody was used (HMB-45: 790-4366, Ventana validated protocol).

    Techniques:

    Effect of reference genes on the calculated expression of Irf4 and Zbtb20 .The relative expression of Irf4 ( a ) and Zbtb20 ( b ) in aLN and iLN were normalized with Actb , Ywhaz , Gapdh , Tbp , Ubc , Hprt or Hmbs by RT-qPCR after 1 and 7 days of the 1st and 3rd vaccination (V1D1, V1D7, V3D1 and V3D7, respectively). Data are presented as mean + SEM. Comparison of ΔΔCq values by one-way ANOVA and Tukey’s multiple comparison test was performed. Experimental groups identified with different symbols were significantly different from each other ( P

    Journal: BMC Research Notes

    Article Title: Selection of a suitable reference gene for quantitative gene expression in mouse lymph nodes after vaccination

    doi: 10.1186/s13104-017-3005-y

    Figure Lengend Snippet: Effect of reference genes on the calculated expression of Irf4 and Zbtb20 .The relative expression of Irf4 ( a ) and Zbtb20 ( b ) in aLN and iLN were normalized with Actb , Ywhaz , Gapdh , Tbp , Ubc , Hprt or Hmbs by RT-qPCR after 1 and 7 days of the 1st and 3rd vaccination (V1D1, V1D7, V3D1 and V3D7, respectively). Data are presented as mean + SEM. Comparison of ΔΔCq values by one-way ANOVA and Tukey’s multiple comparison test was performed. Experimental groups identified with different symbols were significantly different from each other ( P

    Article Snippet: Quantitative real time PCR Real time qPCR was performed in a total volume of 10 µl using iTaq™ Universal Probes Supermix (2× concentrated master mix) (Bio-Rad) and primer/probe sets from Taqman® Gene Express Assay (Applied Biosystems, Waltham, MA, USA) with 5′-FAM TaqMan® MGB probe and 3′-nonfluorescent quencher for genes including Hprt (Mm03024075_m1), Hmbs (Mm01143545_m1), Gapdh (Mm99999915_g1), Ubc (Mm01201237_m1), β-actin (Actb, Mm00607939_s1), Tbp (Mm01277045_m1), Ywhaz (Mm03950126_s1), interferon regulatory factor 4 (Irf4 , Mm00516431_m1), interleukin 4 (Il4, Mm00445259_m1), zinc finger and BTB domain-containing protein 20 (Zbtb20 , Mm00457765_m1) and tumor necrosis factor receptor superfamily member 17 (Tnfrsf17 , Mm00495683_m1).

    Techniques: Expressing, Quantitative RT-PCR

    Effect of reference genes on the calculated expression of Tnfrsf17 and Il4 . The relative expression of Tnfrsf17 ( a ) and Il4 ( b ) in aLN and iLN were normalized with Actb , Ywhaz , Gapdh , Tbp , Ubc , Hprt or Hmbs by RT-qPCR after 1 and 7 days of the 1st and 3rd vaccination (V1D1, V1D7, V3D1 and V3D7, respectively). Data are presented as mean + SEM. Comparison of ΔΔCq values by one-way ANOVA and Tukey’s multiple comparison test was performed. Experimental groups identified with different symbols were significantly different from each other ( P

    Journal: BMC Research Notes

    Article Title: Selection of a suitable reference gene for quantitative gene expression in mouse lymph nodes after vaccination

    doi: 10.1186/s13104-017-3005-y

    Figure Lengend Snippet: Effect of reference genes on the calculated expression of Tnfrsf17 and Il4 . The relative expression of Tnfrsf17 ( a ) and Il4 ( b ) in aLN and iLN were normalized with Actb , Ywhaz , Gapdh , Tbp , Ubc , Hprt or Hmbs by RT-qPCR after 1 and 7 days of the 1st and 3rd vaccination (V1D1, V1D7, V3D1 and V3D7, respectively). Data are presented as mean + SEM. Comparison of ΔΔCq values by one-way ANOVA and Tukey’s multiple comparison test was performed. Experimental groups identified with different symbols were significantly different from each other ( P

    Article Snippet: Quantitative real time PCR Real time qPCR was performed in a total volume of 10 µl using iTaq™ Universal Probes Supermix (2× concentrated master mix) (Bio-Rad) and primer/probe sets from Taqman® Gene Express Assay (Applied Biosystems, Waltham, MA, USA) with 5′-FAM TaqMan® MGB probe and 3′-nonfluorescent quencher for genes including Hprt (Mm03024075_m1), Hmbs (Mm01143545_m1), Gapdh (Mm99999915_g1), Ubc (Mm01201237_m1), β-actin (Actb, Mm00607939_s1), Tbp (Mm01277045_m1), Ywhaz (Mm03950126_s1), interferon regulatory factor 4 (Irf4 , Mm00516431_m1), interleukin 4 (Il4, Mm00445259_m1), zinc finger and BTB domain-containing protein 20 (Zbtb20 , Mm00457765_m1) and tumor necrosis factor receptor superfamily member 17 (Tnfrsf17 , Mm00495683_m1).

    Techniques: Expressing, Quantitative RT-PCR

    Decrease of furin activity delays tumor growth of Rh30 xenografts in vivo A. Schematic overview of Rh30 xenograft model. 3-4 NOD/Scid mice per group were fed with or without DOX-supplemented food starting 7 days prior engraftment. Mice were then engrafted s.c. with 2.5 million Rh30 cells. Tumor growth was monitored over time and mice were sacrificed once a tumor reached a size of 1000 mm 3 . B. Absence of furin activity delays initial tumor growth. Tumor growth rate was monitored by caliper measurements. Data represent mean tumor size of 3-4 mice per group. C. Furin expression is reduced upon induction of furin specific shRNA. Tumor tissue was collected upon sacrifice of mice, total RNA isolated and furin mRNA levels analyzed by qRT-PCR. Expression levels relative to HMBS are shown.

    Journal: Oncotarget

    Article Title: The proprotein convertase furin is required to maintain viability of alveolar rhabdomyosarcoma cells

    doi: 10.18632/oncotarget.11648

    Figure Lengend Snippet: Decrease of furin activity delays tumor growth of Rh30 xenografts in vivo A. Schematic overview of Rh30 xenograft model. 3-4 NOD/Scid mice per group were fed with or without DOX-supplemented food starting 7 days prior engraftment. Mice were then engrafted s.c. with 2.5 million Rh30 cells. Tumor growth was monitored over time and mice were sacrificed once a tumor reached a size of 1000 mm 3 . B. Absence of furin activity delays initial tumor growth. Tumor growth rate was monitored by caliper measurements. Data represent mean tumor size of 3-4 mice per group. C. Furin expression is reduced upon induction of furin specific shRNA. Tumor tissue was collected upon sacrifice of mice, total RNA isolated and furin mRNA levels analyzed by qRT-PCR. Expression levels relative to HMBS are shown.

    Article Snippet: Cycle threshold (CT ) values were normalized to GAPDH (Hs02758991_g1) or HMBS (Hs00609296_g1) for cells or tumor tissue, respectively.

    Techniques: Activity Assay, In Vivo, Mouse Assay, Expressing, shRNA, Isolation, Quantitative RT-PCR

    HMB-45 expression in normal Langerhans cells without skin damage caused by melanoma (magnification, ×400).

    Journal: Biomedical Reports

    Article Title: Expression of microphthalmia transcription factor, S100 protein, and HMB-45 in malignant melanoma and pigmented nevi

    doi: 10.3892/br.2016.732

    Figure Lengend Snippet: HMB-45 expression in normal Langerhans cells without skin damage caused by melanoma (magnification, ×400).

    Article Snippet: Furthermore, it was not expressed in other components of skin tissue, which was similar to HMB-45.

    Techniques: Expressing

    Expression of S100 protein, HMB-45, and MITF in MM and pigmented nevus samples. (A) S100 protein in MM (+++; magnification, ×200); (B) HMB-45 in MM (magnification, ×400); (C) HMB-45 in Spitz nevus (+++; magnification, ×200); (D) MITF in MM (+; magnification, ×100); (E) MITF in metastatic MM (+; magnification, ×100); (F) MITF in Spitz nevus (+; magnification, ×100). MITF, microphthalmia transcription factor; MM, malignant melanoma.

    Journal: Biomedical Reports

    Article Title: Expression of microphthalmia transcription factor, S100 protein, and HMB-45 in malignant melanoma and pigmented nevi

    doi: 10.3892/br.2016.732

    Figure Lengend Snippet: Expression of S100 protein, HMB-45, and MITF in MM and pigmented nevus samples. (A) S100 protein in MM (+++; magnification, ×200); (B) HMB-45 in MM (magnification, ×400); (C) HMB-45 in Spitz nevus (+++; magnification, ×200); (D) MITF in MM (+; magnification, ×100); (E) MITF in metastatic MM (+; magnification, ×100); (F) MITF in Spitz nevus (+; magnification, ×100). MITF, microphthalmia transcription factor; MM, malignant melanoma.

    Article Snippet: Furthermore, it was not expressed in other components of skin tissue, which was similar to HMB-45.

    Techniques: Expressing

    Absence of GDNF modulation by estradiol replacement in ovariectomized female mice. (A) Mouse experimental model. Ovariectomized (ovx) animals were separated in four groups and received either sham implant (O) or E 2 implant (Oi, E1 and E5) at surgery time. Three weeks later, mice received Vehicle (O and Oi), a single s.c. E 2 injection (E1), or five E 2 injections over 5 days (E5). Animals were sacrificed 24 hours after s.c. injection (O, Oi, E1) or 4–5 hours after the last injection (E5). (B) Vertical bar graph showing the mean ± SEM of plasma E 2 in O, Oi, E1 and E5 mice. This shows that the estrogen replacement protocol induced an increase in circulating E 2 in ovx mice. (C) Western blot analysis of phosphorylated Akt (pAkt) showing a representative blot (left), and pAkt / Akt ratio is shown with a vertical bar graph (right). The greatest difference is found between O and E1 (p = 0.053). (D) Gdnf mRNA expression, reported as Gdnf1-2 relative to bActin , in the striatum of wild-type ( Gdnf +/+ ) and heterozygous mice ( Gdnf +/- ). (E) GDNF protein content expressed as pg / mg total protein in Gdnf +/+ and Gdnf +/- striatum. (F) Scatter plot diagram showing the positive correlation (ρ = 0.72, p = 0.002) between Gdnf mRNA (shown in D) and GDNF protein levels (shown in E) in Gdnf +/+ and Gdnf +/- striata. This data shows that GDNF protein concentration measured by ELISA is consistent with Gdnf gene expression level. (G) QPCR analysis of Gdnf expression measured by Gdnf1-2 relative to Parv mRNA (left graph) or Hmbs mRNA (right). (H) ELISA analysis of GDNF protein levels showing no difference between the experimental groups. All data are presented as the mean ± SEM. *, p

    Journal: PLoS ONE

    Article Title: Striatal GDNF Production Is Independent to Circulating Estradiol Level Despite Pan-Neuronal Activation in the Female Mouse

    doi: 10.1371/journal.pone.0164391

    Figure Lengend Snippet: Absence of GDNF modulation by estradiol replacement in ovariectomized female mice. (A) Mouse experimental model. Ovariectomized (ovx) animals were separated in four groups and received either sham implant (O) or E 2 implant (Oi, E1 and E5) at surgery time. Three weeks later, mice received Vehicle (O and Oi), a single s.c. E 2 injection (E1), or five E 2 injections over 5 days (E5). Animals were sacrificed 24 hours after s.c. injection (O, Oi, E1) or 4–5 hours after the last injection (E5). (B) Vertical bar graph showing the mean ± SEM of plasma E 2 in O, Oi, E1 and E5 mice. This shows that the estrogen replacement protocol induced an increase in circulating E 2 in ovx mice. (C) Western blot analysis of phosphorylated Akt (pAkt) showing a representative blot (left), and pAkt / Akt ratio is shown with a vertical bar graph (right). The greatest difference is found between O and E1 (p = 0.053). (D) Gdnf mRNA expression, reported as Gdnf1-2 relative to bActin , in the striatum of wild-type ( Gdnf +/+ ) and heterozygous mice ( Gdnf +/- ). (E) GDNF protein content expressed as pg / mg total protein in Gdnf +/+ and Gdnf +/- striatum. (F) Scatter plot diagram showing the positive correlation (ρ = 0.72, p = 0.002) between Gdnf mRNA (shown in D) and GDNF protein levels (shown in E) in Gdnf +/+ and Gdnf +/- striata. This data shows that GDNF protein concentration measured by ELISA is consistent with Gdnf gene expression level. (G) QPCR analysis of Gdnf expression measured by Gdnf1-2 relative to Parv mRNA (left graph) or Hmbs mRNA (right). (H) ELISA analysis of GDNF protein levels showing no difference between the experimental groups. All data are presented as the mean ± SEM. *, p

    Article Snippet: For quantitative PCR, we used the following TaqMan probes (from Thermofisher): Gdnf1-2 (Gdnf exons 1 to 2), Mm00599849_m1; Pvalb (parvalbumin), Mm00443100_m1; Actb (βActin), Mm00443100_m1; Gapdh , Mm99999915_g1; Hmbs , Mm00660262_g1.

    Techniques: Mouse Assay, Injection, Western Blot, Expressing, Protein Concentration, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

    mRNA quantification in amphetamine and saline treated animals. Normalized quantities for D 1 , D 2 and PDE10A acquired in amphetamine and saline treated. GUSB and HMBS were used for the normalization of quantities values. Data are presented as mean ± SD. No significant difference in mRNA levels of amphetamine and saline treated animals could be found

    Journal: Ejnmmi Radiopharmacy and Chemistry

    Article Title: Striatal phosphodiesterase 10A availability is altered secondary to chronic changes in dopamine neurotransmission

    doi: 10.1186/s41181-016-0005-5

    Figure Lengend Snippet: mRNA quantification in amphetamine and saline treated animals. Normalized quantities for D 1 , D 2 and PDE10A acquired in amphetamine and saline treated. GUSB and HMBS were used for the normalization of quantities values. Data are presented as mean ± SD. No significant difference in mRNA levels of amphetamine and saline treated animals could be found

    Article Snippet: A qPCR core kit without dUTP (Eurogentec, Seraing, Belgium) was combined, according to protocol, with pre-designed Taqman Gene Expression Assays (Applied Biosystems) to quantify the genes of interest; PDE10A (Rn00673152_m1), D1 receptor (Rn03062203_s1) and D2 receptor (Rn00561126_m1) and internal control genes corresponding to glucuronidase b (GUSB, Rn00566655_m1), hydroxymethylbilane synthase (HMBS, Rn00565886_m1), phosphoglycerate kinase 1 (PGK1, Rn00821429_g1), peptidylpropyl isomerase b (PPIB, Rn03302274_m1) and transferrin receptor (TFRC, Rn01474701_m1, all Applied Biosystems).

    Techniques:

    Immunohistochemical stains showing cytoplasmic positivity to HMB-45 (2A ×400) and melan A (2B ×400).

    Journal: Annals of Saudi Medicine

    Article Title: Mimicry of sugar tumor and minute pulmonary meningothelial-like nodule to metastatic lung deposits in a patient with rectal adenocarcinoma

    doi: 10.5144/0256-4947.2013.400

    Figure Lengend Snippet: Immunohistochemical stains showing cytoplasmic positivity to HMB-45 (2A ×400) and melan A (2B ×400).

    Article Snippet: The neoplastic cells showed membranous positivity to CD34 and vimentin (mouse monoclonal, ready to use, Novocastra), cytoplasmic positivity to HMB-45 (mouse monoclonal, 1:50, Novocastra) and melan A (mouse monoclonal, 1:25, Novocastra) in most of the cells ( , ) and focal nuclear and cytoplasmic positivity to S100 (rabbit polyclonal, ready to use, Novocastra).

    Techniques: Immunohistochemistry

    Immunohistochemical stains showing cytoplasmic positivity to HMB-45 (2A ×400) and melan A (2B ×400).

    Journal: Annals of Saudi Medicine

    Article Title: Mimicry of sugar tumor and minute pulmonary meningothelial-like nodule to metastatic lung deposits in a patient with rectal adenocarcinoma

    doi: 10.5144/0256-4947.2013.400

    Figure Lengend Snippet: Immunohistochemical stains showing cytoplasmic positivity to HMB-45 (2A ×400) and melan A (2B ×400).

    Article Snippet: The neoplastic cells showed membranous positivity to CD34 and vimentin (mouse monoclonal, ready to use, Novocastra), cytoplasmic positivity to HMB-45 (mouse monoclonal, 1:50, Novocastra) and melan A (mouse monoclonal, 1:25, Novocastra) in most of the cells ( , ) and focal nuclear and cytoplasmic positivity to S100 (rabbit polyclonal, ready to use, Novocastra).

    Techniques: Immunohistochemistry

    Linearity of radial, axial, and current stiffness of HMB from COMSOL (gray color) and MBTR (black color) on Geometry 6, the optimized HMB. Radial stiffness: −5.1 (MBTR) and −5.9 (FEA) N/mm, Axial stiffness 3.1 (MBTR) and 3.2 (FEA) N/mm,

    Journal: Mechatronics : the science of intelligent machines

    Article Title: Optimization of a Hybrid Magnetic Bearing for a Magnetically Levitated Blood Pump via 3-D FEA

    doi: 10.1016/j.mechatronics.2011.07.010

    Figure Lengend Snippet: Linearity of radial, axial, and current stiffness of HMB from COMSOL (gray color) and MBTR (black color) on Geometry 6, the optimized HMB. Radial stiffness: −5.1 (MBTR) and −5.9 (FEA) N/mm, Axial stiffness 3.1 (MBTR) and 3.2 (FEA) N/mm,

    Article Snippet: In order to find out the magnetic flux loop of the HMB caused by the coil current, the magnetic flux distribution of the revised HMB were simulated and visualized in COMSOL with 0A and with 1A coil current applied on the y direction. is generated in the absence of the coil current; the two points A and B denote the air gap between the small stator iron and small rotor iron on the y direction and C and D denote the air gap between large irons.

    Techniques:

    Screening of CYP2W1 inducers in the colon adenocarcinoma cell line HCC2998. CYP2W1 mRNA levels following the 48 h treatment with several selected drugs. The CYP2W1 transcript expression was normalized by housekeeping genes EIF2B2, HMBS and PPIA and related to control (vehicle alone, either DMSO or ethanol depending on the drug). Data are presented as mean±SEM of at least two experiments in at least two replicates per experiment. * p

    Journal: PLoS ONE

    Article Title: Developmental Regulation and Induction of Cytochrome P450 2W1, an Enzyme Expressed in Colon Tumors

    doi: 10.1371/journal.pone.0122820

    Figure Lengend Snippet: Screening of CYP2W1 inducers in the colon adenocarcinoma cell line HCC2998. CYP2W1 mRNA levels following the 48 h treatment with several selected drugs. The CYP2W1 transcript expression was normalized by housekeeping genes EIF2B2, HMBS and PPIA and related to control (vehicle alone, either DMSO or ethanol depending on the drug). Data are presented as mean±SEM of at least two experiments in at least two replicates per experiment. * p

    Article Snippet: An internal control sample on the plate and housekeeping genes, EIF2B2 (Hs00204540_m1), HMBS (Hs00609293_g1) and PPIA (Hs99999904_m1) for human and Tjp1 (Mm00493699_m1), 18S (Mm03928990_g1) and Gadph (4352339E) for mice were used for normalization according to the 2-ΔΔCt method [ ].

    Techniques: Expressing

    Immunohistochemical staining showing (a) Positivity of tumor cells with S-100 and negativity for (b) Pan cytokeratin, (c) HMB 45, and (d) Vimentin (×400)

    Journal: Journal of Oral and Maxillofacial Pathology : JOMFP

    Article Title: Malignant peripheral nerve sheath tumor of facial nerve: Presenting as parotid mass

    doi: 10.4103/0973-029X.110708

    Figure Lengend Snippet: Immunohistochemical staining showing (a) Positivity of tumor cells with S-100 and negativity for (b) Pan cytokeratin, (c) HMB 45, and (d) Vimentin (×400)

    Article Snippet: Immunohistochemically, the tumor cells showed diffuse and strong positivity for S-100, (Dako), however, were negative for Pan-cytokeratin (Dako, AE1/AE3), Desmin (Dako, D33), and Human Melanoma Black (HMB) 45 (Dako) [ ] and Vimentin (Dako).

    Techniques: Immunohistochemistry, Staining