beta naphthoflavone β nf  (Millipore)

 
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    Name:
    Betulinic acid
    Description:
    Betulinic acid is a lupane type triterpene which can be prepared from betulin via oxidation using chromium oxide VI Along with anti HIV 1 and anti tumor activity it also shows other bioactivities such as anti inflammatory antibacterial in vitro antimalarial anthelmintic and antioxidant activities
    Catalog Number:
    855057
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    Structured Review

    Millipore beta naphthoflavone β nf
    Betulinic acid
    Betulinic acid is a lupane type triterpene which can be prepared from betulin via oxidation using chromium oxide VI Along with anti HIV 1 and anti tumor activity it also shows other bioactivities such as anti inflammatory antibacterial in vitro antimalarial anthelmintic and antioxidant activities
    https://www.bioz.com/result/beta naphthoflavone β nf/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    beta naphthoflavone β nf - by Bioz Stars, 2021-07
    97/100 stars

    Images

    1) Product Images from "Detection of Cytochrome P450 mRNA in Tissue Sections and Cell Lines Using Enzyme-Labeled Fluorescence In Situ Hybridization"

    Article Title: Detection of Cytochrome P450 mRNA in Tissue Sections and Cell Lines Using Enzyme-Labeled Fluorescence In Situ Hybridization

    Journal: In vitro toxicology

    doi:

    Enzyme-labeled fluorescence (ELF) in situ hybridization control experiments utilizing cultured rat hepatoma cells and formalin-fixed, paraffin-embedded human tissue sections. (A) 22-μM beta-napthoflavone (β-NF)-induced H4IIE rat cells hybridized with rat biotin-conjugated CYP1A1 oligomer. (B) 22-μM β-NF-treated H4IIe rat cells probed with the rat biotin-labeled CYP1A1 20-mer and pretreated with 150 μ/ml of RNase A. (C) 22-μM β-NF-exposed H4IIE rat cells hybridized with rat biotinylated CYP1A1 2Gmer plus 60-fold excess nonbiotinylated rat CYP1A1 probe. (D) Poorly differentiated squamous cell carcinoma of human oral epithelium probed with human CYP1A1 biotin-labeled 20-mer. (E) Poorly differentiated squamous cell carcinoma of human oral epithelium hybridized with the partially homologous rat CYP1A1 biotin-conjugated oligomer probe. (F) Poorly differentiated squamous cell carcinoma of human oral epithelium without the addition of a biotinylated oligomer to the hybridization buffer. Magnification 400×.
    Figure Legend Snippet: Enzyme-labeled fluorescence (ELF) in situ hybridization control experiments utilizing cultured rat hepatoma cells and formalin-fixed, paraffin-embedded human tissue sections. (A) 22-μM beta-napthoflavone (β-NF)-induced H4IIE rat cells hybridized with rat biotin-conjugated CYP1A1 oligomer. (B) 22-μM β-NF-treated H4IIe rat cells probed with the rat biotin-labeled CYP1A1 20-mer and pretreated with 150 μ/ml of RNase A. (C) 22-μM β-NF-exposed H4IIE rat cells hybridized with rat biotinylated CYP1A1 2Gmer plus 60-fold excess nonbiotinylated rat CYP1A1 probe. (D) Poorly differentiated squamous cell carcinoma of human oral epithelium probed with human CYP1A1 biotin-labeled 20-mer. (E) Poorly differentiated squamous cell carcinoma of human oral epithelium hybridized with the partially homologous rat CYP1A1 biotin-conjugated oligomer probe. (F) Poorly differentiated squamous cell carcinoma of human oral epithelium without the addition of a biotinylated oligomer to the hybridization buffer. Magnification 400×.

    Techniques Used: Labeling, Fluorescence, In Situ Hybridization, Cell Culture, Formalin-fixed Paraffin-Embedded, Hybridization

    Enzyme-labeled fluorescence (ELF) in situ hybridization using a rat biotin-conjugated CYP1A1 oligomer probe with cultured rat hepatoma cells. (A9 untreated H4IIE cells probed with biotinylated CYP1A1 oligomer. (B) 22-μM beta-naphoflavone (β-NF)-treated H4IIE cells hybridized with biotin-labeled CYP1A1 oligomer (C) 22-μM β-NF exposed H4IIE cells lacking the biotinylated CYP1A1 probe from the hybridization solution. (D) Unexposed Fao cells probed with biotinylated CYP1A1 oligomer. (E) 22-μM β-NF-treated Fao cells hybridized with biotin-labeled CYP1A1 oligomer (F) 22-μM β-NF-exposed Fao cells without the biotinylated CYP1A1 probe. Magnification 400×.
    Figure Legend Snippet: Enzyme-labeled fluorescence (ELF) in situ hybridization using a rat biotin-conjugated CYP1A1 oligomer probe with cultured rat hepatoma cells. (A9 untreated H4IIE cells probed with biotinylated CYP1A1 oligomer. (B) 22-μM beta-naphoflavone (β-NF)-treated H4IIE cells hybridized with biotin-labeled CYP1A1 oligomer (C) 22-μM β-NF exposed H4IIE cells lacking the biotinylated CYP1A1 probe from the hybridization solution. (D) Unexposed Fao cells probed with biotinylated CYP1A1 oligomer. (E) 22-μM β-NF-treated Fao cells hybridized with biotin-labeled CYP1A1 oligomer (F) 22-μM β-NF-exposed Fao cells without the biotinylated CYP1A1 probe. Magnification 400×.

    Techniques Used: Labeling, Fluorescence, In Situ Hybridization, Cell Culture, Hybridization

    Related Articles

    Staining:

    Article Title: Functional recovery and neural differentiation after transplantation of allogenic adipose-derived stem cells in a canine model of acute spinal cord injury
    Article Snippet: Adipogenic differentiation: ASCs were initially cultured and propagated up to 80~90% confluence in K-NAC medium containing 5% FBS and then shifted to adipogenic medium [DMEM high-glucose medium with 10% FBS, 10 µg/mL insulin (Sigma-Aldrich, USA), 1 µM dexamethasone (Sigma-Aldrich, USA), 0.2 mM indomethacin (Sigma-Aldrich, USA), and 0.5 mM isobutylmethylxanthine (Sigma-Aldrich, USA)] for 3 days, then to DMEM high-glucose medium with 10% FBS, 10 µg/mL insulin (Sigma-Aldrich, USA) for 4 days. .. The accumulation of neutral lipids was detected by staining ASCs in a solution of 0.5% Oil red O. Osteogenic differentiation: ASCs were initially cultured and propagated up to 70% confluence in K-NAC medium containing 5% FBS and then shifted to osteogenic medium [DMEM low-glucose medium with 10% FBS, 0.1 µM dexamethasone (Sigma-Aldrich, USA), 50 µM l-Ascorbate-2-phosphate (Sigma-Aldrich, USA), and 10 mM beta-glycerophosphate (Sigma-Aldrich, USA)] for 3 weeks [ ]. .. Mineralization was assessed by staining ASCs with 40 mM Alizarin red S (pH 4.1).

    Cell Culture:

    Article Title: Functional recovery and neural differentiation after transplantation of allogenic adipose-derived stem cells in a canine model of acute spinal cord injury
    Article Snippet: Adipogenic differentiation: ASCs were initially cultured and propagated up to 80~90% confluence in K-NAC medium containing 5% FBS and then shifted to adipogenic medium [DMEM high-glucose medium with 10% FBS, 10 µg/mL insulin (Sigma-Aldrich, USA), 1 µM dexamethasone (Sigma-Aldrich, USA), 0.2 mM indomethacin (Sigma-Aldrich, USA), and 0.5 mM isobutylmethylxanthine (Sigma-Aldrich, USA)] for 3 days, then to DMEM high-glucose medium with 10% FBS, 10 µg/mL insulin (Sigma-Aldrich, USA) for 4 days. .. The accumulation of neutral lipids was detected by staining ASCs in a solution of 0.5% Oil red O. Osteogenic differentiation: ASCs were initially cultured and propagated up to 70% confluence in K-NAC medium containing 5% FBS and then shifted to osteogenic medium [DMEM low-glucose medium with 10% FBS, 0.1 µM dexamethasone (Sigma-Aldrich, USA), 50 µM l-Ascorbate-2-phosphate (Sigma-Aldrich, USA), and 10 mM beta-glycerophosphate (Sigma-Aldrich, USA)] for 3 weeks [ ]. .. Mineralization was assessed by staining ASCs with 40 mM Alizarin red S (pH 4.1).

    other:

    Article Title: Supported Zeolite Beta Layers via an Organic Template-Free Preparation Route
    Article Snippet: Preparation of the Zeolite Beta Seed Layer on Porous Stainless Steel Supports The zeolite beta seed layers were prepared from a molar gel with a composition of 1 SiO2 :0.56 TEAOH:0.02 Al2 O3 :15 H2 O. Ludox AS40 (40%, Sigma Aldrich, St. Louis, MO, USA) and Al(NO3 )3 ·9 H2 O (98%, Fluka, St. Louis, MO, USA) were used as silica and alumina sources, respectively.

    Western Blot:

    Article Title: Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells
    Article Snippet: .. After western blotting of the egg extract, the membranes were incubated overnight at 4 °C with rabbit polyclonal Xenopus anti-Lamin B3 (1:10 000, a gift from N. Morin, France), Xenopus anti-importin alpha1 (1:5000, a gift from K. Weiss, Switzerland), mouse monoclonal rat anti-karyopherin β1 (1:1000 KPNB1, Antibodies-online, clone 23), and anti-beta actin (1:5000, Sigma, clone AC15). .. Immunolabelling was revealed with Uptima Uptilight HRP Chemiluminescent Substrate (Uptima-Interchim 58372B).

    Incubation:

    Article Title: Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells
    Article Snippet: .. After western blotting of the egg extract, the membranes were incubated overnight at 4 °C with rabbit polyclonal Xenopus anti-Lamin B3 (1:10 000, a gift from N. Morin, France), Xenopus anti-importin alpha1 (1:5000, a gift from K. Weiss, Switzerland), mouse monoclonal rat anti-karyopherin β1 (1:1000 KPNB1, Antibodies-online, clone 23), and anti-beta actin (1:5000, Sigma, clone AC15). .. Immunolabelling was revealed with Uptima Uptilight HRP Chemiluminescent Substrate (Uptima-Interchim 58372B).

    Article Title: Human-Induced Pluripotent Stem Cells Manufactured Using a Current Good Manufacturing Practice-Compliant Process Differentiate Into Clinically Relevant Cells From Three Germ Layers
    Article Snippet: After rinsing the fixed cells with PBS-Tween solution, the cells were incubated with DPBS containing 10% goat serum (Life Technologies, 10000C) for 2 h at room temperature. .. Primary antibodies detecting alpha-1 Fetoprotein (Abcam, ab3980; 1:200 or R & D systems, MAB1369, 1:100), beta III tubulin (Millipore, MAB1637; 1:400), and Smooth Muscle Actin (DAKO, M0851; 1:500) were added to blocked cultures and incubated overnight at 2–8°C. .. The cells were rinsed twice with PBS-T, and the secondary antibody, Alexa Fluor 494-conjugated goat anti-mouse IgG(H + L) (Life Technologies, A-11032; 1:1,000) or Alexa Fluor 488-conjugated goat anti-mouse IgG(H + L) (Life Technologies, A11001; 1:1,000) were added and incubated for at least 2 h at room temperature.

    Article Title: Evidence for Gardnerella vaginalis uptake and internalization by squamous vaginal epithelial cells: implications for the pathogenesis of bacterial vaginosis
    Article Snippet: .. Cells were then air dried for 15 min, hydrated in Tris saline (50 mM Tris, 150 mM NaCl) pH 7.4 for 5 min and incubated at 37 °C for 1 h with a mixture of a rabbit polyclonal antibody to estrogen receptor beta at 1:100 dilution (Millipore Billerica, MA) and a monoclonal antibody to a surface protein (described above) used to identify G. vaginalis at 1:50 dilution. .. Cells were then washed 3 times with Tris saline and incubated at 37 °C for 30 min with a mixture of secondary donkey anti-rabbit antibodies conjugated to rhodamine, and donkey anti-mouse antibodies conjugated to FITC (Jackson ImmunoResearch, West Grove, PA), both at a 1:100 dilution in PBS (pH 7.4).

    Plasmid Purification:

    Article Title: Beta-4 tubulin identifies a primitive cell source for oligodendrocytes in the mammalian brain
    Article Snippet: Staining of BrdU-treated cultures included pretreatment with 2 N HCl (1 hour) and 0.1 M sodium borate, pH 8.6 (30 min) followed by application of FITC-conjugated monoclonal anti-BrdU antibody (1:20, Abcam, Cambridge, MA) overnight at 4°C. .. The following other primary and secondary antibodies and dilutions we used: Class IV beta tubulin (1:15,000, Sigma), Tuj1 (1:200, Covance), GFAP (1:10000, Dako), O4 (1:5, Gift from Dr. Robert Miller), A2B5 (1:40, Gift from Dr. Robert Miller), NG2 (1:100, Chemicon International), PLP (1:1000, Agmed), PSA-NCAM (1:100, Pharmingen, San Diego, CA), goat anti-rat IgG biotinylated (1:1000, Vector Laboratories), goat anti-mouse IgG Alexa (1:1000, Invitrogen), goat anti-rabbit FITC (1:1000, Invitrogen), goat anti-mouse IgM FITC (1:500, Southern Biotechnology Associates, Birmingham, AB), and goat anti-rabbit IgG Alexa (1:1000, Invitrogen). .. DAPI was included in the mounting medium (Vectashield, Vector Laboratories) for nuclear detection.

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  • 93
    Millipore nuclear factor κ b
    Asian dust particles-induced tumor necrosis factor- α (TNF- α ) production is dependent on mitogen-activated protein kinase and nuclear factor- <t>κ</t> B signaling pathways. RAW264.7 cells were pretreated for 30 min with (a) 50 μ M of SP600125, an inhibitor of c-Jun N-terminal kinase; (b) 30 μ M of U0126, an extracellular signal-regulated kinase (ERK) inhibitor; or (c) 50 μ M of <t>SN50,</t> a nuclear factor- κ B (NF- κ B) inhibitor and then treated for 6 h with 100 μ g/mL of ADP1 or ADP2 or 1.5 μ g/mL of LPS. Dimethyl sulfoxide (0.1%) vehicle was used as control (Cont.). The level of TNF- α in the culture supernatants was assessed by means of an enzyme-linked immunosorbent assay. Results are expressed as mean ± SD; n = 6; * P
    Nuclear Factor κ B, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore beta naphthoflavone β nf
    Enzyme-labeled fluorescence (ELF) in situ hybridization control experiments utilizing cultured rat hepatoma cells and formalin-fixed, paraffin-embedded human tissue sections. (A) 22-μM <t>beta-napthoflavone</t> <t>(β-NF)-induced</t> H4IIE rat cells hybridized with rat biotin-conjugated CYP1A1 oligomer. (B) 22-μM β-NF-treated H4IIe rat cells probed with the rat biotin-labeled CYP1A1 20-mer and pretreated with 150 μ/ml of RNase A. (C) 22-μM β-NF-exposed H4IIE rat cells hybridized with rat biotinylated CYP1A1 2Gmer plus 60-fold excess nonbiotinylated rat CYP1A1 probe. (D) Poorly differentiated squamous cell carcinoma of human oral epithelium probed with human CYP1A1 biotin-labeled 20-mer. (E) Poorly differentiated squamous cell carcinoma of human oral epithelium hybridized with the partially homologous rat CYP1A1 biotin-conjugated oligomer probe. (F) Poorly differentiated squamous cell carcinoma of human oral epithelium without the addition of a biotinylated oligomer to the hybridization buffer. Magnification 400×.
    Beta Naphthoflavone β Nf, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/beta naphthoflavone β nf/product/Millipore
    Average 97 stars, based on 1 article reviews
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    93
    Millipore nuclear factor kappa b nf κb
    rMIC1 and rMIC4 induce IL-12 production by macrophages through activation of TGF-β activated kinase 1 (TAK1), p38, and nuclear factor-kappa B (NF-κB). ( A ) RAW264.7- luc  murine macrophages were stimulated with the following preparations of  Toxoplasma gondii  microneme proteins: rMIC1 (5 µg/mL), rMIC4 (5 µg/mL), and the native Lac+ fraction (5 µg/mL), which contains MIC1 and MIC4. LPS (500 ng/mL) and medium were used as the positive and negative controls, respectively. Cells were lysed 4 h post-stimulation, and NF-κB activation was inferred from the luminescence measurements. Data are expressed as the mean ± SD of triplicate wells, and data from three independent experiments yielding similar results. ( B ) Total lysates of bone marrow-derived macrophages (BMDMs) was were 30 min after stimulation with rMIC1 (5 µg/mL), rMIC4 (5 µg/mL), or LPS (1 µg/mL). As controls, cells were also incubated with medium (i.e., unstimulated cells [US]) or sampled at time zero (i.e., T0), as indicated at the top of the panels. Immunoblotting was performed to assess total p38 and the p65 subunit of NF-κB, as well as their phosphorylated forms (p-p38 and p-NF-κB). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( C , D ) Densitometric analysis to quantify the p38 and NF-κB bands in the Western blot (panel B). ( E , F ) IL-12 concentrations in the supernatant of BMDM cultures that were pretreated with vehicle (-) or pharmacological inhibitors of AKT (Wortmannin, 100 nM), TAK1 (5Z-7-oxozeaenol, 100 nM), ERK (PD9805, 20 μM), JNK (SP600125, 20 μM), p38 (SB202190, 20 μM), or NF-κB (Caffeic acid, 15 µg/mL) for 3 h, and subsequently stimulated with ( E ) rMIC1 (5 µg/mL) or ( F ) rMIC4 (5 µg/mL) for 24 h as assessed by ELISA. LPS (1 µg/mL) and medium were used as the positive and negative controls, respectively. ( G ) Viability of BMDMs pretreated with pharmacological inhibitors of signaling molecules. Statistical analysis comparing the viability of cells (%) that were pretreated with each inhibitor and stimulated with rMIC1 or rMIC4 to that of cells stimulated with rMICs but not pretreated with pharmacological inhibitors (vehicle). Staurosporine (2 µM) and Triton X-100 (1%  v / v ) were used to induce in vitro apoptosis. The data are from two independent experiments yielding similar results. (*)  p
    Nuclear Factor Kappa B Nf κb, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore β naphthoflavone
    Increased epithelial proliferation correlates with increased cell fusion. (A) Schematic representation of experimental design. AhCre + ;Apc fl/fl mice were transplanted with GFP-expressing whole bone marrow (WBM) on day 0. Two days later, Cre recombinase was induced by <t>β-naphthoflavone</t> (β-NF) administration for 4 consecutive days (days 2–5). Mice were sacrificed on day 7 and the distal small intestine analyzed for fusion (B–E). The intestinal-specific deletion of Apc resulted in an extensive hyperproliferation of crypt cells compared to wild-type (WT) mice, as seen by H E stain (C vs. B) and Ki67 staining (red, indicated by yellow brackets; E vs. D). (F–K) Detection of GFP-expressing cells (green; yellow arrowheads mark examples) denoting cell fusion was increased in the AhCre + ;Apc −/− mice compared to mock-injected WT mice. Three patterns of cell fusion were observed: (G) crypt-only, (H) villus-only, (I) both crypt and villus regions in one crypt/villus unit. Panel (J) is a higher magnification of the red box in panel (I) demonstrating that the Paneth cell region at the base of the crypt remained GFP-negative. Solid white lines denote epithelial/luminal border; dashed white lines indicate epithelial/mesenchymal border. Bars = 25 µm. (K) A significant increase in fusion was observed in villus only ( P = 0.011) and crypt/villus ( P = 0.009) AhCre + ;Apc −/− mice (gray bars) compared to mock-injected WT mice (black bars).
    β Naphthoflavone, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Asian dust particles-induced tumor necrosis factor- α (TNF- α ) production is dependent on mitogen-activated protein kinase and nuclear factor- κ B signaling pathways. RAW264.7 cells were pretreated for 30 min with (a) 50 μ M of SP600125, an inhibitor of c-Jun N-terminal kinase; (b) 30 μ M of U0126, an extracellular signal-regulated kinase (ERK) inhibitor; or (c) 50 μ M of SN50, a nuclear factor- κ B (NF- κ B) inhibitor and then treated for 6 h with 100 μ g/mL of ADP1 or ADP2 or 1.5 μ g/mL of LPS. Dimethyl sulfoxide (0.1%) vehicle was used as control (Cont.). The level of TNF- α in the culture supernatants was assessed by means of an enzyme-linked immunosorbent assay. Results are expressed as mean ± SD; n = 6; * P

    Journal: Journal of Immunology Research

    Article Title: Asian Dust Particles Induce Macrophage Inflammatory Responses via Mitogen-Activated Protein Kinase Activation and Reactive Oxygen Species Production

    doi: 10.1155/2014/856154

    Figure Lengend Snippet: Asian dust particles-induced tumor necrosis factor- α (TNF- α ) production is dependent on mitogen-activated protein kinase and nuclear factor- κ B signaling pathways. RAW264.7 cells were pretreated for 30 min with (a) 50 μ M of SP600125, an inhibitor of c-Jun N-terminal kinase; (b) 30 μ M of U0126, an extracellular signal-regulated kinase (ERK) inhibitor; or (c) 50 μ M of SN50, a nuclear factor- κ B (NF- κ B) inhibitor and then treated for 6 h with 100 μ g/mL of ADP1 or ADP2 or 1.5 μ g/mL of LPS. Dimethyl sulfoxide (0.1%) vehicle was used as control (Cont.). The level of TNF- α in the culture supernatants was assessed by means of an enzyme-linked immunosorbent assay. Results are expressed as mean ± SD; n = 6; * P

    Article Snippet: SN50, a nuclear factor-κ B (NF-κ B) inhibitor, was purchased from Calbiochem (La Jolla, CA).

    Techniques: Enzyme-linked Immunosorbent Assay

    Enzyme-labeled fluorescence (ELF) in situ hybridization control experiments utilizing cultured rat hepatoma cells and formalin-fixed, paraffin-embedded human tissue sections. (A) 22-μM beta-napthoflavone (β-NF)-induced H4IIE rat cells hybridized with rat biotin-conjugated CYP1A1 oligomer. (B) 22-μM β-NF-treated H4IIe rat cells probed with the rat biotin-labeled CYP1A1 20-mer and pretreated with 150 μ/ml of RNase A. (C) 22-μM β-NF-exposed H4IIE rat cells hybridized with rat biotinylated CYP1A1 2Gmer plus 60-fold excess nonbiotinylated rat CYP1A1 probe. (D) Poorly differentiated squamous cell carcinoma of human oral epithelium probed with human CYP1A1 biotin-labeled 20-mer. (E) Poorly differentiated squamous cell carcinoma of human oral epithelium hybridized with the partially homologous rat CYP1A1 biotin-conjugated oligomer probe. (F) Poorly differentiated squamous cell carcinoma of human oral epithelium without the addition of a biotinylated oligomer to the hybridization buffer. Magnification 400×.

    Journal: In vitro toxicology

    Article Title: Detection of Cytochrome P450 mRNA in Tissue Sections and Cell Lines Using Enzyme-Labeled Fluorescence In Situ Hybridization

    doi:

    Figure Lengend Snippet: Enzyme-labeled fluorescence (ELF) in situ hybridization control experiments utilizing cultured rat hepatoma cells and formalin-fixed, paraffin-embedded human tissue sections. (A) 22-μM beta-napthoflavone (β-NF)-induced H4IIE rat cells hybridized with rat biotin-conjugated CYP1A1 oligomer. (B) 22-μM β-NF-treated H4IIe rat cells probed with the rat biotin-labeled CYP1A1 20-mer and pretreated with 150 μ/ml of RNase A. (C) 22-μM β-NF-exposed H4IIE rat cells hybridized with rat biotinylated CYP1A1 2Gmer plus 60-fold excess nonbiotinylated rat CYP1A1 probe. (D) Poorly differentiated squamous cell carcinoma of human oral epithelium probed with human CYP1A1 biotin-labeled 20-mer. (E) Poorly differentiated squamous cell carcinoma of human oral epithelium hybridized with the partially homologous rat CYP1A1 biotin-conjugated oligomer probe. (F) Poorly differentiated squamous cell carcinoma of human oral epithelium without the addition of a biotinylated oligomer to the hybridization buffer. Magnification 400×.

    Article Snippet: Beta-naphthoflavone (β-NF) was purchased from Aldrich Chemical Co. (Milwaukee, WI), and levamisole was acquired from Sigma Chemical Co. (St. Louis, MO).

    Techniques: Labeling, Fluorescence, In Situ Hybridization, Cell Culture, Formalin-fixed Paraffin-Embedded, Hybridization

    Enzyme-labeled fluorescence (ELF) in situ hybridization using a rat biotin-conjugated CYP1A1 oligomer probe with cultured rat hepatoma cells. (A9 untreated H4IIE cells probed with biotinylated CYP1A1 oligomer. (B) 22-μM beta-naphoflavone (β-NF)-treated H4IIE cells hybridized with biotin-labeled CYP1A1 oligomer (C) 22-μM β-NF exposed H4IIE cells lacking the biotinylated CYP1A1 probe from the hybridization solution. (D) Unexposed Fao cells probed with biotinylated CYP1A1 oligomer. (E) 22-μM β-NF-treated Fao cells hybridized with biotin-labeled CYP1A1 oligomer (F) 22-μM β-NF-exposed Fao cells without the biotinylated CYP1A1 probe. Magnification 400×.

    Journal: In vitro toxicology

    Article Title: Detection of Cytochrome P450 mRNA in Tissue Sections and Cell Lines Using Enzyme-Labeled Fluorescence In Situ Hybridization

    doi:

    Figure Lengend Snippet: Enzyme-labeled fluorescence (ELF) in situ hybridization using a rat biotin-conjugated CYP1A1 oligomer probe with cultured rat hepatoma cells. (A9 untreated H4IIE cells probed with biotinylated CYP1A1 oligomer. (B) 22-μM beta-naphoflavone (β-NF)-treated H4IIE cells hybridized with biotin-labeled CYP1A1 oligomer (C) 22-μM β-NF exposed H4IIE cells lacking the biotinylated CYP1A1 probe from the hybridization solution. (D) Unexposed Fao cells probed with biotinylated CYP1A1 oligomer. (E) 22-μM β-NF-treated Fao cells hybridized with biotin-labeled CYP1A1 oligomer (F) 22-μM β-NF-exposed Fao cells without the biotinylated CYP1A1 probe. Magnification 400×.

    Article Snippet: Beta-naphthoflavone (β-NF) was purchased from Aldrich Chemical Co. (Milwaukee, WI), and levamisole was acquired from Sigma Chemical Co. (St. Louis, MO).

    Techniques: Labeling, Fluorescence, In Situ Hybridization, Cell Culture, Hybridization

    rMIC1 and rMIC4 induce IL-12 production by macrophages through activation of TGF-β activated kinase 1 (TAK1), p38, and nuclear factor-kappa B (NF-κB). ( A ) RAW264.7- luc  murine macrophages were stimulated with the following preparations of  Toxoplasma gondii  microneme proteins: rMIC1 (5 µg/mL), rMIC4 (5 µg/mL), and the native Lac+ fraction (5 µg/mL), which contains MIC1 and MIC4. LPS (500 ng/mL) and medium were used as the positive and negative controls, respectively. Cells were lysed 4 h post-stimulation, and NF-κB activation was inferred from the luminescence measurements. Data are expressed as the mean ± SD of triplicate wells, and data from three independent experiments yielding similar results. ( B ) Total lysates of bone marrow-derived macrophages (BMDMs) was were 30 min after stimulation with rMIC1 (5 µg/mL), rMIC4 (5 µg/mL), or LPS (1 µg/mL). As controls, cells were also incubated with medium (i.e., unstimulated cells [US]) or sampled at time zero (i.e., T0), as indicated at the top of the panels. Immunoblotting was performed to assess total p38 and the p65 subunit of NF-κB, as well as their phosphorylated forms (p-p38 and p-NF-κB). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( C , D ) Densitometric analysis to quantify the p38 and NF-κB bands in the Western blot (panel B). ( E , F ) IL-12 concentrations in the supernatant of BMDM cultures that were pretreated with vehicle (-) or pharmacological inhibitors of AKT (Wortmannin, 100 nM), TAK1 (5Z-7-oxozeaenol, 100 nM), ERK (PD9805, 20 μM), JNK (SP600125, 20 μM), p38 (SB202190, 20 μM), or NF-κB (Caffeic acid, 15 µg/mL) for 3 h, and subsequently stimulated with ( E ) rMIC1 (5 µg/mL) or ( F ) rMIC4 (5 µg/mL) for 24 h as assessed by ELISA. LPS (1 µg/mL) and medium were used as the positive and negative controls, respectively. ( G ) Viability of BMDMs pretreated with pharmacological inhibitors of signaling molecules. Statistical analysis comparing the viability of cells (%) that were pretreated with each inhibitor and stimulated with rMIC1 or rMIC4 to that of cells stimulated with rMICs but not pretreated with pharmacological inhibitors (vehicle). Staurosporine (2 µM) and Triton X-100 (1%  v / v ) were used to induce in vitro apoptosis. The data are from two independent experiments yielding similar results. (*)  p

    Journal: International Journal of Molecular Sciences

    Article Title: Receptor Heterodimerization and Co-Receptor Engagement in TLR2 Activation Induced by MIC1 and MIC4 from Toxoplasma gondii

    doi: 10.3390/ijms20205001

    Figure Lengend Snippet: rMIC1 and rMIC4 induce IL-12 production by macrophages through activation of TGF-β activated kinase 1 (TAK1), p38, and nuclear factor-kappa B (NF-κB). ( A ) RAW264.7- luc murine macrophages were stimulated with the following preparations of Toxoplasma gondii microneme proteins: rMIC1 (5 µg/mL), rMIC4 (5 µg/mL), and the native Lac+ fraction (5 µg/mL), which contains MIC1 and MIC4. LPS (500 ng/mL) and medium were used as the positive and negative controls, respectively. Cells were lysed 4 h post-stimulation, and NF-κB activation was inferred from the luminescence measurements. Data are expressed as the mean ± SD of triplicate wells, and data from three independent experiments yielding similar results. ( B ) Total lysates of bone marrow-derived macrophages (BMDMs) was were 30 min after stimulation with rMIC1 (5 µg/mL), rMIC4 (5 µg/mL), or LPS (1 µg/mL). As controls, cells were also incubated with medium (i.e., unstimulated cells [US]) or sampled at time zero (i.e., T0), as indicated at the top of the panels. Immunoblotting was performed to assess total p38 and the p65 subunit of NF-κB, as well as their phosphorylated forms (p-p38 and p-NF-κB). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( C , D ) Densitometric analysis to quantify the p38 and NF-κB bands in the Western blot (panel B). ( E , F ) IL-12 concentrations in the supernatant of BMDM cultures that were pretreated with vehicle (-) or pharmacological inhibitors of AKT (Wortmannin, 100 nM), TAK1 (5Z-7-oxozeaenol, 100 nM), ERK (PD9805, 20 μM), JNK (SP600125, 20 μM), p38 (SB202190, 20 μM), or NF-κB (Caffeic acid, 15 µg/mL) for 3 h, and subsequently stimulated with ( E ) rMIC1 (5 µg/mL) or ( F ) rMIC4 (5 µg/mL) for 24 h as assessed by ELISA. LPS (1 µg/mL) and medium were used as the positive and negative controls, respectively. ( G ) Viability of BMDMs pretreated with pharmacological inhibitors of signaling molecules. Statistical analysis comparing the viability of cells (%) that were pretreated with each inhibitor and stimulated with rMIC1 or rMIC4 to that of cells stimulated with rMICs but not pretreated with pharmacological inhibitors (vehicle). Staurosporine (2 µM) and Triton X-100 (1% v / v ) were used to induce in vitro apoptosis. The data are from two independent experiments yielding similar results. (*) p

    Article Snippet: Cell Signaling Inhibition Assay BMDMs were harvested from C57BL/6 mice, plated into 24-well plates at 5 × 105 cells/well, and incubated for 3 h with the following pharmacological inhibitors of MAP-kinases: TGF-β activated kinase 1 (TAK1), 5Z-7-oxozeaenol, 100 nM; extracellular-signal-regulated kinase (ERK), PD98059, 20 μM; c-Jun N-terminal kinase (JNK), SP600125, 20 μM; p38, SB202190, 20 μM; Ser and Thr kinase (AKT), Wortmannin, 100 nM; nuclear factor-kappa B (NF-κB), and caffeic acid, 15 µg/mL (Sigma-Aldrich).

    Techniques: Activation Assay, Derivative Assay, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, In Vitro

    Increased epithelial proliferation correlates with increased cell fusion. (A) Schematic representation of experimental design. AhCre + ;Apc fl/fl mice were transplanted with GFP-expressing whole bone marrow (WBM) on day 0. Two days later, Cre recombinase was induced by β-naphthoflavone (β-NF) administration for 4 consecutive days (days 2–5). Mice were sacrificed on day 7 and the distal small intestine analyzed for fusion (B–E). The intestinal-specific deletion of Apc resulted in an extensive hyperproliferation of crypt cells compared to wild-type (WT) mice, as seen by H E stain (C vs. B) and Ki67 staining (red, indicated by yellow brackets; E vs. D). (F–K) Detection of GFP-expressing cells (green; yellow arrowheads mark examples) denoting cell fusion was increased in the AhCre + ;Apc −/− mice compared to mock-injected WT mice. Three patterns of cell fusion were observed: (G) crypt-only, (H) villus-only, (I) both crypt and villus regions in one crypt/villus unit. Panel (J) is a higher magnification of the red box in panel (I) demonstrating that the Paneth cell region at the base of the crypt remained GFP-negative. Solid white lines denote epithelial/luminal border; dashed white lines indicate epithelial/mesenchymal border. Bars = 25 µm. (K) A significant increase in fusion was observed in villus only ( P = 0.011) and crypt/villus ( P = 0.009) AhCre + ;Apc −/− mice (gray bars) compared to mock-injected WT mice (black bars).

    Journal: PLoS ONE

    Article Title: Inflammation and Proliferation Act Together to Mediate Intestinal Cell Fusion

    doi: 10.1371/journal.pone.0006530

    Figure Lengend Snippet: Increased epithelial proliferation correlates with increased cell fusion. (A) Schematic representation of experimental design. AhCre + ;Apc fl/fl mice were transplanted with GFP-expressing whole bone marrow (WBM) on day 0. Two days later, Cre recombinase was induced by β-naphthoflavone (β-NF) administration for 4 consecutive days (days 2–5). Mice were sacrificed on day 7 and the distal small intestine analyzed for fusion (B–E). The intestinal-specific deletion of Apc resulted in an extensive hyperproliferation of crypt cells compared to wild-type (WT) mice, as seen by H E stain (C vs. B) and Ki67 staining (red, indicated by yellow brackets; E vs. D). (F–K) Detection of GFP-expressing cells (green; yellow arrowheads mark examples) denoting cell fusion was increased in the AhCre + ;Apc −/− mice compared to mock-injected WT mice. Three patterns of cell fusion were observed: (G) crypt-only, (H) villus-only, (I) both crypt and villus regions in one crypt/villus unit. Panel (J) is a higher magnification of the red box in panel (I) demonstrating that the Paneth cell region at the base of the crypt remained GFP-negative. Solid white lines denote epithelial/luminal border; dashed white lines indicate epithelial/mesenchymal border. Bars = 25 µm. (K) A significant increase in fusion was observed in villus only ( P = 0.011) and crypt/villus ( P = 0.009) AhCre + ;Apc −/− mice (gray bars) compared to mock-injected WT mice (black bars).

    Article Snippet: AhCre+ ; Apcfl/fl progeny were induced by intraperitoneal injection of β-naphthoflavone (β-NF; Sigma) dissolved in corn oil (80 mg/kg) for four days and analyzed 2 days later.

    Techniques: Mouse Assay, Expressing, Staining, Injection