monensin  (Millipore)

 
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  • 99
    Name:
    Monensin sodium salt
    Description:
    Chemical structure polyether
    Catalog Number:
    m5273
    Price:
    None
    Applications:
    Monensin sodium salt has been used:. for the intracellular staining of cytokines. to study its effects on the regulation of nuclear factor-κ B (NF-κB ) activation. as a positive control to study its effects on in vitro rumen fermentation
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    Structured Review

    Millipore monensin
    Monensin sodium salt
    Chemical structure polyether
    https://www.bioz.com/result/monensin/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    monensin - by Bioz Stars, 2021-03
    99/100 stars

    Images

    1) Product Images from "The Antibiotic Monensin Causes Cell Cycle Disruption of Toxoplasma gondii Mediated through the DNA Repair Enzyme TgMSH-1 ▿"

    Article Title: The Antibiotic Monensin Causes Cell Cycle Disruption of Toxoplasma gondii Mediated through the DNA Repair Enzyme TgMSH-1 ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01092-10

    Monensin causes changes in transcriptional regulation of many T. gondii genes, including upregulation of canonical histones.
    Figure Legend Snippet: Monensin causes changes in transcriptional regulation of many T. gondii genes, including upregulation of canonical histones.

    Techniques Used:

    Model for TgMSH-1-dependent monensin action on T. gondii . In this model, monensin results directly or indirectly in damage to mitochondrial DNA or another undetermined stress. This results in a TgMSH-1-mediated signal transduction cascade, possibly involving
    Figure Legend Snippet: Model for TgMSH-1-dependent monensin action on T. gondii . In this model, monensin results directly or indirectly in damage to mitochondrial DNA or another undetermined stress. This results in a TgMSH-1-mediated signal transduction cascade, possibly involving

    Techniques Used: Transduction

    Venn diagram showing overlap of genes that are transcriptionally upregulated in G 1 and S/M phases of the T. gondii ) and the 30 most strongly transcriptionally upregulated genes in T. gondii exposed to monensin. Values in parentheses indicate
    Figure Legend Snippet: Venn diagram showing overlap of genes that are transcriptionally upregulated in G 1 and S/M phases of the T. gondii ) and the 30 most strongly transcriptionally upregulated genes in T. gondii exposed to monensin. Values in parentheses indicate

    Techniques Used:

    Monensin causes changes in transcriptional regulation of many T. gondii genes, including upregulation of canonical histones.
    Figure Legend Snippet: Monensin causes changes in transcriptional regulation of many T. gondii genes, including upregulation of canonical histones.

    Techniques Used:

    Flow cytometry analysis of parental (RH) and TgMSH1-deficient ( Tgmsh-1 mut ) T. gondii strains in response to monensin. Intracellular parasites were exposed to either normal culture medium or culture medium plus 0.75 ng monensin/ml for 24 h. DNA content
    Figure Legend Snippet: Flow cytometry analysis of parental (RH) and TgMSH1-deficient ( Tgmsh-1 mut ) T. gondii strains in response to monensin. Intracellular parasites were exposed to either normal culture medium or culture medium plus 0.75 ng monensin/ml for 24 h. DNA content

    Techniques Used: Flow Cytometry, Cytometry

    Monensin causes changes in transcriptional regulation of many T. gondii genes, including upregulation of canonical histones.
    Figure Legend Snippet: Monensin causes changes in transcriptional regulation of many T. gondii genes, including upregulation of canonical histones.

    Techniques Used:

    Effect of PI-3-K family (PX-866) and ATM kinase (KU-55933) inhibitors on monensin-induced cell cycle redistribution. Intracellular parasites were exposed either to normal culture medium or culture medium plus 5μΜ PX-866 or 2μΜ
    Figure Legend Snippet: Effect of PI-3-K family (PX-866) and ATM kinase (KU-55933) inhibitors on monensin-induced cell cycle redistribution. Intracellular parasites were exposed either to normal culture medium or culture medium plus 5μΜ PX-866 or 2μΜ

    Techniques Used:

    2) Product Images from "Role for Influenza Virus Envelope Cholesterol in Virus Entry and Infection"

    Article Title: Role for Influenza Virus Envelope Cholesterol in Virus Entry and Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.23.12543-12551.2003

    Envelope cholesterol depletion inhibits influenza virus fusion. (A) Influenza virus was pretreated with 20 mM methyl-β-cyclodextrin or was left untreated and was bound to the surfaces of MDBK cells on ice. Fusion at the cell surface was induced by exposure to pH 5.0 buffer (a to d), or control samples were treated with pH 7.3 buffer (e and f). All samples were treated with monensin to block endocytosis. The cells were analyzed by indirect immunofluorescence microscopy at 5 h postinfection using anti-NP antibodies (b, d, and f), or the nuclei were stained with Hoechst 33258 (a, c, and e). (B) Quantitation of data in panel A with various concentrations of methyl-β-cyclodextrin. At least 100 cells were counted for each sample to determine the percentages of infectivity. The error bars represent the standard errors of the mean. (C) Influenza virus was pretreated with 20 mM methyl-β-cyclodextrin or was left untreated and was bound to the surfaces of MDBK cells on ice. Fusion at the cell surface was induced by exposure to pH 5.0 buffer. All samples were treated with monensin to block endocytosis. The cells were analyzed by FACS analysis using anti-NP antibodies. Negative control, uninfected cells. (D) Bar chart of FACS analysis as described for panel C with various concentrations of methyl-β-cyclodextrin.
    Figure Legend Snippet: Envelope cholesterol depletion inhibits influenza virus fusion. (A) Influenza virus was pretreated with 20 mM methyl-β-cyclodextrin or was left untreated and was bound to the surfaces of MDBK cells on ice. Fusion at the cell surface was induced by exposure to pH 5.0 buffer (a to d), or control samples were treated with pH 7.3 buffer (e and f). All samples were treated with monensin to block endocytosis. The cells were analyzed by indirect immunofluorescence microscopy at 5 h postinfection using anti-NP antibodies (b, d, and f), or the nuclei were stained with Hoechst 33258 (a, c, and e). (B) Quantitation of data in panel A with various concentrations of methyl-β-cyclodextrin. At least 100 cells were counted for each sample to determine the percentages of infectivity. The error bars represent the standard errors of the mean. (C) Influenza virus was pretreated with 20 mM methyl-β-cyclodextrin or was left untreated and was bound to the surfaces of MDBK cells on ice. Fusion at the cell surface was induced by exposure to pH 5.0 buffer. All samples were treated with monensin to block endocytosis. The cells were analyzed by FACS analysis using anti-NP antibodies. Negative control, uninfected cells. (D) Bar chart of FACS analysis as described for panel C with various concentrations of methyl-β-cyclodextrin.

    Techniques Used: Blocking Assay, Immunofluorescence, Microscopy, Staining, Quantitation Assay, Infection, FACS, Negative Control

    3) Product Images from "Stimulation of Cell Proliferation by Endosomal Epidermal Growth Factor Receptor As Revealed through Two Distinct Phases of Signaling"

    Article Title: Stimulation of Cell Proliferation by Endosomal Epidermal Growth Factor Receptor As Revealed through Two Distinct Phases of Signaling

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.16.5803-5815.2003

    Schematics of the two treatment assays employed. (A) Schematic of the continuous and discontinuous EGF stimulation assays employed. For discontinuous treatment, “w” indicates washout of unbound ligand, and solid arrows imply a continuation of signaling (from internalized receptors) after growth factor is removed from the medium. (B) Schematic of the (discontinuous) endosome-associated EGFR stimulation assay employed. For each endosome EGF pulse, the gray bar represents preincubation with AG1478, the red bar indicates incubation with EGF with or without monensin, and “w” indicates washout of AG1478 and thus the onset of endosome EGFR activation. Solid arrows imply the extent of actively signaling EGFR following this wash step.
    Figure Legend Snippet: Schematics of the two treatment assays employed. (A) Schematic of the continuous and discontinuous EGF stimulation assays employed. For discontinuous treatment, “w” indicates washout of unbound ligand, and solid arrows imply a continuation of signaling (from internalized receptors) after growth factor is removed from the medium. (B) Schematic of the (discontinuous) endosome-associated EGFR stimulation assay employed. For each endosome EGF pulse, the gray bar represents preincubation with AG1478, the red bar indicates incubation with EGF with or without monensin, and “w” indicates washout of AG1478 and thus the onset of endosome EGFR activation. Solid arrows imply the extent of actively signaling EGFR following this wash step.

    Techniques Used: Incubation, Activation Assay

    Two pulses of endosomal EGFR signaling are sufficient to stimulate cell proliferation. Proliferation was induced from two temporally separate endosomal EGF pulses. MDCK and BT20 cells were plated at 10,000 cells per coverslip, starved for 36 h, and then treated with AG1478 (AG) and EGF (100 ng/ml), with or without monensin (Mon), followed by washing and incubation in starvation medium. Some cells were left until the end of the assay, while others were treated as described above a second time for 4, 8, or 12 h. Cells treated without monensin were instead stripped of plasma membrane-recycled ligand prior to EGFR activation. At 18 h, cells were fixed and assayed for BrdU incorporation. Cells were counted at 300 per sample, and data were plotted as the mean of triplicate experiments.
    Figure Legend Snippet: Two pulses of endosomal EGFR signaling are sufficient to stimulate cell proliferation. Proliferation was induced from two temporally separate endosomal EGF pulses. MDCK and BT20 cells were plated at 10,000 cells per coverslip, starved for 36 h, and then treated with AG1478 (AG) and EGF (100 ng/ml), with or without monensin (Mon), followed by washing and incubation in starvation medium. Some cells were left until the end of the assay, while others were treated as described above a second time for 4, 8, or 12 h. Cells treated without monensin were instead stripped of plasma membrane-recycled ligand prior to EGFR activation. At 18 h, cells were fixed and assayed for BrdU incorporation. Cells were counted at 300 per sample, and data were plotted as the mean of triplicate experiments.

    Techniques Used: Incubation, Activation Assay, BrdU Incorporation Assay

    4) Product Images from "Rules of chemokine receptor association with T cell polarization in vivo"

    Article Title: Rules of chemokine receptor association with T cell polarization in vivo

    Journal: Journal of Clinical Investigation

    doi:

    CKR association with T cell polarization in inflammatory tissues (CD4 T cells in psoriatic arthritic synovial fluid). ( a ) CKR expression by synovial CD4 T cells. ( b ) CD45RA and CD45RO expression by synovial CD4 T cells. ( c ) Coexpression of CXCR3 and CCR4 by synovial fluid CD4 T cells. ( d ) Intracellular IFN-γ and IL-4 production pattern of arthritic synovial CD4 T cells. ( e ) Frequencies of Th1 (IFN-γ + IL-4 – ), Th2 (IL-4 + IFN-γ – ), Th0 (IFN-γ + IL-4 + ), or Tnp (IFN-γ – IL-4 – ) cells in CKR-expressing CD4 T cell populations are shown. ( f ) Expression of CKRs by Th1, Th2, Th0, and Tnp cells in arthritis synovial fluid. Prestained cells with anti-CKR and anti-CD4 were activated by PMA and ionomycin for 4 hours in the presence of monensin before intracellular staining of IFN-γ and IL-4. Representative data from three different experiments are shown.
    Figure Legend Snippet: CKR association with T cell polarization in inflammatory tissues (CD4 T cells in psoriatic arthritic synovial fluid). ( a ) CKR expression by synovial CD4 T cells. ( b ) CD45RA and CD45RO expression by synovial CD4 T cells. ( c ) Coexpression of CXCR3 and CCR4 by synovial fluid CD4 T cells. ( d ) Intracellular IFN-γ and IL-4 production pattern of arthritic synovial CD4 T cells. ( e ) Frequencies of Th1 (IFN-γ + IL-4 – ), Th2 (IL-4 + IFN-γ – ), Th0 (IFN-γ + IL-4 + ), or Tnp (IFN-γ – IL-4 – ) cells in CKR-expressing CD4 T cell populations are shown. ( f ) Expression of CKRs by Th1, Th2, Th0, and Tnp cells in arthritis synovial fluid. Prestained cells with anti-CKR and anti-CD4 were activated by PMA and ionomycin for 4 hours in the presence of monensin before intracellular staining of IFN-γ and IL-4. Representative data from three different experiments are shown.

    Techniques Used: Expressing, Staining

    5) Product Images from "Analgesia linked to Nav1.7 loss of function requires µ- and δ-opioid receptors"

    Article Title: Analgesia linked to Nav1.7 loss of function requires µ- and δ-opioid receptors

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.14687.1

    Both Penk and Nfat5 expression are regulated by intracellular sodium concentration. ( A ) Penk and Nfat5 expression levels in cultured DRG neurons treated with monensin (500 nM, 30 and 60 min, respectively, light and dark blue bars). Control neurons (white bar) were treated with vehicle (ethanol) for 60 min. ( B ) Penk and Nfat5 mRNA quantification in cultured DRG neurons treated with tetrodotoxin (TTX) (500 nM, 6 h). Control neurons received same volume of saline solution for 6 h (red bar). ( C ) Penk and ( D ) Nfat5 transcripts levels in wild-type (WT) compared to Nav1.7 knockout (KO) DRG neurons treated by TTX (500 nM, 6h). TTX induced Penk overexpression is correlated with Nfat5 expression level, both are dependant of Nav1.7. ( E ) Penk and ( F ) Nfat5 expression in WT and Nav1.7 KO cultured DRG neurons treated with veratridine (1 µM, 6h). Results are presented as mean ± SEM. Data were analysed by two-way ANOVA followed by the Bonferroni post hoc test. * p
    Figure Legend Snippet: Both Penk and Nfat5 expression are regulated by intracellular sodium concentration. ( A ) Penk and Nfat5 expression levels in cultured DRG neurons treated with monensin (500 nM, 30 and 60 min, respectively, light and dark blue bars). Control neurons (white bar) were treated with vehicle (ethanol) for 60 min. ( B ) Penk and Nfat5 mRNA quantification in cultured DRG neurons treated with tetrodotoxin (TTX) (500 nM, 6 h). Control neurons received same volume of saline solution for 6 h (red bar). ( C ) Penk and ( D ) Nfat5 transcripts levels in wild-type (WT) compared to Nav1.7 knockout (KO) DRG neurons treated by TTX (500 nM, 6h). TTX induced Penk overexpression is correlated with Nfat5 expression level, both are dependant of Nav1.7. ( E ) Penk and ( F ) Nfat5 expression in WT and Nav1.7 KO cultured DRG neurons treated with veratridine (1 µM, 6h). Results are presented as mean ± SEM. Data were analysed by two-way ANOVA followed by the Bonferroni post hoc test. * p

    Techniques Used: Expressing, Concentration Assay, Cell Culture, Knock-Out, Over Expression

    6) Product Images from "Penetratin tandemly linked to a CTL peptide induces anti-tumour T-cell responses via a cross-presentation pathway"

    Article Title: Penetratin tandemly linked to a CTL peptide induces anti-tumour T-cell responses via a cross-presentation pathway

    Journal:

    doi: 10.1111/j.1365-2567.2005.02304.x

    IntSIIN is endocytosed and processed in endosomes. DC were incubated for 45 min with inhibitors (a) chloroquine/NH 4 CL (200, 20 μ m ), and (b) monensin (1000, 100 μ m ) followed by incubation with IntSIIN at 1 μg/ml and added to B3Z
    Figure Legend Snippet: IntSIIN is endocytosed and processed in endosomes. DC were incubated for 45 min with inhibitors (a) chloroquine/NH 4 CL (200, 20 μ m ), and (b) monensin (1000, 100 μ m ) followed by incubation with IntSIIN at 1 μg/ml and added to B3Z

    Techniques Used: Incubation

    7) Product Images from "Dedifferentiation of human articular chondrocytes is associated with alterations in expression patterns of GDF-5 and its receptors"

    Article Title: Dedifferentiation of human articular chondrocytes is associated with alterations in expression patterns of GDF-5 and its receptors

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2009.00953.x

    Confocal laser micrographs of dedifferentiated chondrocytes cultivated as monolayer on chamber slides (Alexa 488 in green, propidium iodide nuclear staining in red). Monensin was used at a concentration of 2 μM to enhance the detection of newly synthesized proteins. Scale bar = 10 μm. (a, b) GDF-5: Cells treated without monensin (a) and with 2 μM monensin (b) show little difference with regard to protein distribution. (c, d) BMPR-Ib: BMPR-Ib shows intense staining of the cell membrane (c). Cells treated with 2 μM monensin (d) show vesicles filled with BMPR-Ib, indicating a high synthesis rate of the receptor. (e, f) Double staining of GDF-5 (green) and BMPR-Ib (red) in chondrocytes without monensin (e) and with 2 μM monensin treatment (f).
    Figure Legend Snippet: Confocal laser micrographs of dedifferentiated chondrocytes cultivated as monolayer on chamber slides (Alexa 488 in green, propidium iodide nuclear staining in red). Monensin was used at a concentration of 2 μM to enhance the detection of newly synthesized proteins. Scale bar = 10 μm. (a, b) GDF-5: Cells treated without monensin (a) and with 2 μM monensin (b) show little difference with regard to protein distribution. (c, d) BMPR-Ib: BMPR-Ib shows intense staining of the cell membrane (c). Cells treated with 2 μM monensin (d) show vesicles filled with BMPR-Ib, indicating a high synthesis rate of the receptor. (e, f) Double staining of GDF-5 (green) and BMPR-Ib (red) in chondrocytes without monensin (e) and with 2 μM monensin treatment (f).

    Techniques Used: Staining, Concentration Assay, Synthesized, Double Staining

    Confocal laser micrographs of dedifferentiated chondrocytes cultivated as monolayer on chamber slides (Alexa 488 in green, propidium iodide nuclear staining in red). Monensin was used at a concentration of 2 μM to enhance the detection of newly synthesized proteins. Scale bar = 10 μm. (a, b) Connexin 43: Connexin 43 appears equally distributed at the cell borders (a). When 2 μM monensin is used, newly synthesised connexin 43 remains within the perinuclear vesicles (b). (c, d) Noggin: Noggin is mainly located in the perinuclear area (c). Cells treated with 2 μM monensin show increased vesicle formation containing throughout the cytoplasm (d).
    Figure Legend Snippet: Confocal laser micrographs of dedifferentiated chondrocytes cultivated as monolayer on chamber slides (Alexa 488 in green, propidium iodide nuclear staining in red). Monensin was used at a concentration of 2 μM to enhance the detection of newly synthesized proteins. Scale bar = 10 μm. (a, b) Connexin 43: Connexin 43 appears equally distributed at the cell borders (a). When 2 μM monensin is used, newly synthesised connexin 43 remains within the perinuclear vesicles (b). (c, d) Noggin: Noggin is mainly located in the perinuclear area (c). Cells treated with 2 μM monensin show increased vesicle formation containing throughout the cytoplasm (d).

    Techniques Used: Staining, Concentration Assay, Synthesized

    8) Product Images from "Expression of Neurotrophin-3 (NT-3) and Anterograde Axonal Transport of Endogenous NT-3 by Retinal Ganglion Cells in Chick Embryos"

    Article Title: Expression of Neurotrophin-3 (NT-3) and Anterograde Axonal Transport of Endogenous NT-3 by Retinal Ganglion Cells in Chick Embryos

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.20-02-00736.2000

    Synopsis of NT-3 immunolabel and neurotrophin receptor immunolabel in the superficial layers of the optic tectum in 16-d-old chick embryos and their sources from within the tectum or retinal projection. Shaded ( gray ) areas indicate neuropil label, graded from light (low levels) to dark (high levels). Labeled cell bodies are indicated by dots or profiles (when dendritic details are labeled). +MON , Monensin added in the eye (to reveal nonretinal source of label; intraocular monensin prevents anterograde axonal transport and causes degeneration of RGC axons and axon terminals). The right - hand panel depicts the fiber course of retinal ganglion cell axons in the SO and the SGFS and the morphology of tectal neurons in sublayer SGFSi with ascending dendrites into SGFSg, d, and c.
    Figure Legend Snippet: Synopsis of NT-3 immunolabel and neurotrophin receptor immunolabel in the superficial layers of the optic tectum in 16-d-old chick embryos and their sources from within the tectum or retinal projection. Shaded ( gray ) areas indicate neuropil label, graded from light (low levels) to dark (high levels). Labeled cell bodies are indicated by dots or profiles (when dendritic details are labeled). +MON , Monensin added in the eye (to reveal nonretinal source of label; intraocular monensin prevents anterograde axonal transport and causes degeneration of RGC axons and axon terminals). The right - hand panel depicts the fiber course of retinal ganglion cell axons in the SO and the SGFS and the morphology of tectal neurons in sublayer SGFSi with ascending dendrites into SGFSg, d, and c.

    Techniques Used: Immunolabeling, Labeling

    Effects of intraocular monensin, colchicine, and NT-3 antibodies on retinotectal projections in chick embryos. A , DiI application to the fixed retinotectal projection shows DiI label in the SO and SGFSd ( d, arrowheads ). B , Pretreatment with monensin ( +Mon ) in the eye abolishes DiI label in SGFSd and results in diffuse DiI label in the SO. C , The thickness ( arrow ) of the SO and SGFSa–f in the normal tectum [layer SGFSg is indicated ( g )] is shown. D , Pretreatment of the eye with colchicine ( +Col ) reduces the thickness ( arrow ) of the SO and SGFSa–f. E , Quantification of changes in the thickness of the SO + SGFSa–f tectal layers (ipsilateral vehicle control side = 100%) is shown. Note that axotomy, monensin, and colchicine cause a similar reduction in the thickness of the retinorecipient tectal layers, at least in part because of degeneration of retinotectal fibers and terminals. Error bars indicate SEM. The number of independent experiments ( n ) is indicated. a-NT3 , NT-3 antibody; Axo , axotomy; IgG , normal IgG control; Veh , vehicle. Scale bars: A, B ; C, D , 50 μm.
    Figure Legend Snippet: Effects of intraocular monensin, colchicine, and NT-3 antibodies on retinotectal projections in chick embryos. A , DiI application to the fixed retinotectal projection shows DiI label in the SO and SGFSd ( d, arrowheads ). B , Pretreatment with monensin ( +Mon ) in the eye abolishes DiI label in SGFSd and results in diffuse DiI label in the SO. C , The thickness ( arrow ) of the SO and SGFSa–f in the normal tectum [layer SGFSg is indicated ( g )] is shown. D , Pretreatment of the eye with colchicine ( +Col ) reduces the thickness ( arrow ) of the SO and SGFSa–f. E , Quantification of changes in the thickness of the SO + SGFSa–f tectal layers (ipsilateral vehicle control side = 100%) is shown. Note that axotomy, monensin, and colchicine cause a similar reduction in the thickness of the retinorecipient tectal layers, at least in part because of degeneration of retinotectal fibers and terminals. Error bars indicate SEM. The number of independent experiments ( n ) is indicated. a-NT3 , NT-3 antibody; Axo , axotomy; IgG , normal IgG control; Veh , vehicle. Scale bars: A, B ; C, D , 50 μm.

    Techniques Used:

    Sections through the optic tectum of a 16-d-old chick embryo immunolabeled for BDNF. A , Section through the ipsilateral (control) tectum. B , Section through the experimental tectum, contralateral to the eye that was injected with monensin ( MON ) to block anterograde transport and induce RGC axon degeneration in the tectum. Layer SGFSi ( i ) is indicated. Note that there is no appreciable difference in BDNF label between the two sides of the same tissue section. Scale bar, 100 μm.
    Figure Legend Snippet: Sections through the optic tectum of a 16-d-old chick embryo immunolabeled for BDNF. A , Section through the ipsilateral (control) tectum. B , Section through the experimental tectum, contralateral to the eye that was injected with monensin ( MON ) to block anterograde transport and induce RGC axon degeneration in the tectum. Layer SGFSi ( i ) is indicated. Note that there is no appreciable difference in BDNF label between the two sides of the same tissue section. Scale bar, 100 μm.

    Techniques Used: Immunolabeling, Injection, Blocking Assay

    9) Product Images from "Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process"

    Article Title: Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process

    Journal: Journal of Virology

    doi: 10.1128/JVI.01092-16

    HCMV infection is impaired when endosomal acidification is blocked. (A) HS-578T cells were pretreated with NH 4 Cl (50 mM) at 37°C for 60 min, followed by infection for 5.5 h. RNA was extracted, and HCMV UL123 expression was monitored by RT-qPCR and normalized against GAPDH amplified in the same reaction. (B) HS-578T cells were treated with NH 4 Cl at various concentrations. HCMV infection was carried out for 4.5 h, and the cells were washed with low-acid buffer for 3 min to inactivate uninternalized virus and cultured for additional 3 days without NH 4 Cl before FACS analysis. (C) Cell viability was monitored by CytoTox-One assay using the highest dose (100 mM) of NH 4 Cl. (D and E) HS-578T cells (D) and MRC-5 cells (E) were pretreated with NH 4 Cl (50 mM), monensin (25 nM), or bafilomycin A1 (BFLA1) (200 nM). Representative data from 3 independent experiments for each cell type are shown ( P
    Figure Legend Snippet: HCMV infection is impaired when endosomal acidification is blocked. (A) HS-578T cells were pretreated with NH 4 Cl (50 mM) at 37°C for 60 min, followed by infection for 5.5 h. RNA was extracted, and HCMV UL123 expression was monitored by RT-qPCR and normalized against GAPDH amplified in the same reaction. (B) HS-578T cells were treated with NH 4 Cl at various concentrations. HCMV infection was carried out for 4.5 h, and the cells were washed with low-acid buffer for 3 min to inactivate uninternalized virus and cultured for additional 3 days without NH 4 Cl before FACS analysis. (C) Cell viability was monitored by CytoTox-One assay using the highest dose (100 mM) of NH 4 Cl. (D and E) HS-578T cells (D) and MRC-5 cells (E) were pretreated with NH 4 Cl (50 mM), monensin (25 nM), or bafilomycin A1 (BFLA1) (200 nM). Representative data from 3 independent experiments for each cell type are shown ( P

    Techniques Used: Infection, Expressing, Quantitative RT-PCR, Amplification, Cell Culture, FACS

    10) Product Images from "Developmental Regulation of FKBP65"

    Article Title: Developmental Regulation of FKBP65

    Journal: Molecular Biology of the Cell

    doi:

    FKBP65 is a resident protein of the ER. Subconfluent cultures of FBCs were treated for 3 h with either (A) 10 μg/ml brefeldin A + 10 μg/ml ALLN to “hold” the tropoelastin in the fused ER/Golgi compartment (ALLN is needed to prevent tropoelastin degradation), or (B, C) 10 μM monensin to “hold” the tropoelastin in the cis -Golgi cisternae. Cells were then fixed, permeabilized, and double labeled with a monoclonal antibody to tropoelastin (red) and a polyclonal antibody to either (A, B) FKBP65 (green) or (C) Grp94 (green). After treatment with BFA+ALLN, tropoelastin and FKBP65 were colocalized in the fused ER/Golgi apparatus. When tropoelastin was accumulated in the Golgi, however, the two proteins showed distinct patterns of immunolabeling with tropoelastin in the peri-nuclear Golgi region and FKBP65 remaining in the ER. The same distribution of labeling was seen when Grp94 was colocalized with tropoelastin after monensin treatment. Note that the production of tropoelastin by FBCs in culture is density-dependent, thus not all the cells are synthesizing tropoelastin at subconfluency.
    Figure Legend Snippet: FKBP65 is a resident protein of the ER. Subconfluent cultures of FBCs were treated for 3 h with either (A) 10 μg/ml brefeldin A + 10 μg/ml ALLN to “hold” the tropoelastin in the fused ER/Golgi compartment (ALLN is needed to prevent tropoelastin degradation), or (B, C) 10 μM monensin to “hold” the tropoelastin in the cis -Golgi cisternae. Cells were then fixed, permeabilized, and double labeled with a monoclonal antibody to tropoelastin (red) and a polyclonal antibody to either (A, B) FKBP65 (green) or (C) Grp94 (green). After treatment with BFA+ALLN, tropoelastin and FKBP65 were colocalized in the fused ER/Golgi apparatus. When tropoelastin was accumulated in the Golgi, however, the two proteins showed distinct patterns of immunolabeling with tropoelastin in the peri-nuclear Golgi region and FKBP65 remaining in the ER. The same distribution of labeling was seen when Grp94 was colocalized with tropoelastin after monensin treatment. Note that the production of tropoelastin by FBCs in culture is density-dependent, thus not all the cells are synthesizing tropoelastin at subconfluency.

    Techniques Used: Labeling, Immunolabeling

    11) Product Images from "Internalization and Down-Regulation of the ALK Receptor in Neuroblastoma Cell Lines upon Monoclonal Antibodies Treatment"

    Article Title: Internalization and Down-Regulation of the ALK Receptor in Neuroblastoma Cell Lines upon Monoclonal Antibodies Treatment

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033581

    Agonist mAb treatment induced lysosome targeting and antagonist mAb treatment induced cell surface recyling of ALK receptor. A. CHO cells were transiently transfected with construct encoding for ALK WT. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of agonist mAb 46 and transferrin coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of agonist mAb 46 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merge images were mounted thanks to Photoshop software. B. CHO cells were transiently transfected with construct encoding for ALK wild type. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of antagonist mAb 30 and transferring coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of antagonist mAb 30 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merged images were mounted thanks to photophop software. C. Experiment identical to B but with a cell pre-treatment with monensin (50 µM) for 30 min.
    Figure Legend Snippet: Agonist mAb treatment induced lysosome targeting and antagonist mAb treatment induced cell surface recyling of ALK receptor. A. CHO cells were transiently transfected with construct encoding for ALK WT. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of agonist mAb 46 and transferrin coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of agonist mAb 46 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merge images were mounted thanks to Photoshop software. B. CHO cells were transiently transfected with construct encoding for ALK wild type. Cells were pulsed 10 minutes on ice at 4°C with 6 nM of antagonist mAb 30 and transferring coupled to AlexaFluor 546, then chased at 37°C in a time course manner. Direct immunodetection of antagonist mAb 30 were done with anti-mouse secondary antibody coupled to Alexa Fluor 488. Cellular localization of ALK receptors were shown by immunofluorescence using confocal laser scanning microscopy. Merged images were mounted thanks to photophop software. C. Experiment identical to B but with a cell pre-treatment with monensin (50 µM) for 30 min.

    Techniques Used: Transfection, Construct, Immunodetection, Immunofluorescence, Confocal Laser Scanning Microscopy, Software, Transferring

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    other:

    Article Title: Wnt Modulating Agents Inhibit Human Cytomegalovirus Replication
    Article Snippet: Monensin, nigericin, salinomycin, and ganciclovir (GCV) were purchased from Sigma Chemicals (St. Louis, MO).

    Staining:

    Article Title: House dust mite induced allergic airway disease is attenuated in CD11ccreIL-4Rα−/l°x mice
    Article Snippet: .. For intracellular cytokine staining, lung cells were seeded at 2 × 106 cells/well and stimulated at 37 o C for 4 hours with phorbal myristate acetate (Sigma-Aldrich) (50ng/ml), ionomycin (Sigma-Aldrich) (250ng/ml) and monensin (Sigma-Aldrich) (200 μM) in DMEM/10% FCS. .. CD4+ T cells were stained with CD3-biotin with APC labeled streptavidin and CD4 PerCP, fixed and permeabilized, and intracellular cytokines were stained with PE anti-cytokine antibodies and isotype controls (BD Bioscience).

    Expressing:

    Article Title: TIM-4 Identifies IFN-γ–Expressing Proinflammatory B Effector 1 Cells That Promote Tumor and Allograft Rejection
    Article Snippet: The digested tissue was layered onto Lympholyte M and spun at 1500 × g for 20 min. Lymphocytes isolated from the buffy coat were enumerated. .. The cells were plated at 1 × 106 /ml and stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and monensin (1:1000) for 4–5 h (Sigma Aldrich) for phenotypic assessment of infiltrating lymphocytes and their intracellular cytokine expression. .. μMT mice received 2 × 105 B16 cells (grown as above) by tail vein injection.

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    Reduced Th2 cytokine production in CD11c cre IL-4Rα −/lox mice sensitized and challenged with HDM (day 14). ( A ) Cytokine production by lung CD4 + T cells after stimulation with <t>PMA/ionomycin/monensin.</t> ( B ) Cytokine production by mediastinal lymph node cells after restimulation with HDM extract, measured by ELISA. ( C ) HDM-specific antibodies measured in serum by ELISA. Graphs are representative of two experiments. Significant differences are shown between IL-4Rα −/lox PBS and the other groups unless otherwise indicated by brackets. *P
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    Reduced Th2 cytokine production in CD11c cre IL-4Rα −/lox mice sensitized and challenged with HDM (day 14). ( A ) Cytokine production by lung CD4 + T cells after stimulation with PMA/ionomycin/monensin. ( B ) Cytokine production by mediastinal lymph node cells after restimulation with HDM extract, measured by ELISA. ( C ) HDM-specific antibodies measured in serum by ELISA. Graphs are representative of two experiments. Significant differences are shown between IL-4Rα −/lox PBS and the other groups unless otherwise indicated by brackets. *P

    Journal: Scientific Reports

    Article Title: House dust mite induced allergic airway disease is attenuated in CD11ccreIL-4Rα−/l°x mice

    doi: 10.1038/s41598-017-19060-9

    Figure Lengend Snippet: Reduced Th2 cytokine production in CD11c cre IL-4Rα −/lox mice sensitized and challenged with HDM (day 14). ( A ) Cytokine production by lung CD4 + T cells after stimulation with PMA/ionomycin/monensin. ( B ) Cytokine production by mediastinal lymph node cells after restimulation with HDM extract, measured by ELISA. ( C ) HDM-specific antibodies measured in serum by ELISA. Graphs are representative of two experiments. Significant differences are shown between IL-4Rα −/lox PBS and the other groups unless otherwise indicated by brackets. *P

    Article Snippet: For intracellular cytokine staining, lung cells were seeded at 2 × 106 cells/well and stimulated at 37 o C for 4 hours with phorbal myristate acetate (Sigma-Aldrich) (50ng/ml), ionomycin (Sigma-Aldrich) (250ng/ml) and monensin (Sigma-Aldrich) (200 μM) in DMEM/10% FCS.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Enrichment of interleukin (IL)‐10 producing regulatory T cells (T reg ) cells in visceral leishmaniasis (VL) patients at disease site. (a) Enumeration of IL‐10‐producing T reg (CD4 + FoxP3 + IL‐10 + ) cells in bone marrow mononuclear cells (BM MNCs): isolated MNCs from bone marrow of VL patients were cultured for the assessment of IL‐10 production by antigen‐specific T reg cells in response to Leishmania donovani antigen (whole cell lysate; 10 μg/ml). Cells were cultured for 72 h in animal cell culture conditions (incubation at 37°C with 5% CO 2 ). Monensin (Golgi transport inhibitor; 1 mM) was added in the last 6 h of culture. On forward‐ versus side‐scatter gated lymphocytes, the dual population of CD4 + CD25 + was defined and divided further into CD4 + CD25 dim and CD4 + CD25 br T cells. Histogram plot of FoxP3 expressing CD4 + CD25 dim or CD4 + CD25 br (thin red dotted line) was overlaid on FoxP3 fluorescence minus one (FMO) (Fig. 2a, ii: inset). IL‐10 expression was then assessed on CD4 + CD25 br FoxP3 + T cells. For this, IL‐10 expressing CD4 + CD25 br FoxP3 + T cells were overlaid on IL‐10 FMO (Fig. 2, iii and iv). (b) Increased frequency of IL‐10‐producing T reg cells at disease site in VL patients with high parasitic load (HPL): CD4 + FoxP3 + pregated lymphocytes were used for illustration of the IL‐10‐producing T reg population (histogram). T reg ‐specific IL‐10 population was increased significantly in VL patients with HPL in response to whole cell lysate of L. donovani (10 μg/ml) [ n = 10: mean ± standard deviation (s.d.): 24·03 ± 6·31%] compared to the patients with low parasitic load (LPL) ( n = 12: mean ± s.d.: 3·87 ± 0·49%) ( P = 0·002: unpaired t ‐test). Horizontal lines in scatter‐plot depicted as mean value. (c) Significantly increased soluble level of IL‐10 in VL patients with HPL: enzyme‐linked immunosorbent assay (ELISA)‐based soluble level of IL‐10 in BM aspirate of HPL ( n = 11: mean ± s.d.: 208·5 ± 16·09 pg/ml) patients was significantly higher compared with LPL ( n = 13: mean ± s.d.: 106·3 ± 3·44 pg/ml) patients ( P = 0·0001: unpaired t ]

    Journal: Clinical and Experimental Immunology

    Article Title: T cell suppression in the bone marrow of visceral leishmaniasis patients: impact of parasite load

    doi: 10.1111/cei.13074

    Figure Lengend Snippet: Enrichment of interleukin (IL)‐10 producing regulatory T cells (T reg ) cells in visceral leishmaniasis (VL) patients at disease site. (a) Enumeration of IL‐10‐producing T reg (CD4 + FoxP3 + IL‐10 + ) cells in bone marrow mononuclear cells (BM MNCs): isolated MNCs from bone marrow of VL patients were cultured for the assessment of IL‐10 production by antigen‐specific T reg cells in response to Leishmania donovani antigen (whole cell lysate; 10 μg/ml). Cells were cultured for 72 h in animal cell culture conditions (incubation at 37°C with 5% CO 2 ). Monensin (Golgi transport inhibitor; 1 mM) was added in the last 6 h of culture. On forward‐ versus side‐scatter gated lymphocytes, the dual population of CD4 + CD25 + was defined and divided further into CD4 + CD25 dim and CD4 + CD25 br T cells. Histogram plot of FoxP3 expressing CD4 + CD25 dim or CD4 + CD25 br (thin red dotted line) was overlaid on FoxP3 fluorescence minus one (FMO) (Fig. 2a, ii: inset). IL‐10 expression was then assessed on CD4 + CD25 br FoxP3 + T cells. For this, IL‐10 expressing CD4 + CD25 br FoxP3 + T cells were overlaid on IL‐10 FMO (Fig. 2, iii and iv). (b) Increased frequency of IL‐10‐producing T reg cells at disease site in VL patients with high parasitic load (HPL): CD4 + FoxP3 + pregated lymphocytes were used for illustration of the IL‐10‐producing T reg population (histogram). T reg ‐specific IL‐10 population was increased significantly in VL patients with HPL in response to whole cell lysate of L. donovani (10 μg/ml) [ n = 10: mean ± standard deviation (s.d.): 24·03 ± 6·31%] compared to the patients with low parasitic load (LPL) ( n = 12: mean ± s.d.: 3·87 ± 0·49%) ( P = 0·002: unpaired t ‐test). Horizontal lines in scatter‐plot depicted as mean value. (c) Significantly increased soluble level of IL‐10 in VL patients with HPL: enzyme‐linked immunosorbent assay (ELISA)‐based soluble level of IL‐10 in BM aspirate of HPL ( n = 11: mean ± s.d.: 208·5 ± 16·09 pg/ml) patients was significantly higher compared with LPL ( n = 13: mean ± s.d.: 106·3 ± 3·44 pg/ml) patients ( P = 0·0001: unpaired t ]

    Article Snippet: Monensin (Golgi transport inhibitor; 1 mM; Sigma‐Aldrich) was added in the last 6 h of culture.

    Techniques: Isolation, Cell Culture, Incubation, Expressing, Fluorescence, Standard Deviation, Enzyme-linked Immunosorbent Assay

    Decreased frequency of interferon (IFN)‐γ‐ and interleukin (IL)‐17A‐producing cells in visceral leishmaniasis (VL) patients with high parasitic load (HPL). Enumeration of IFN‐γ‐ and IL‐17A‐producing cells in bone marrow mononuclear cells (BM MNCs): isolated MNCs ( n = 24) from bone marrow of VL patients were cultured for IFN‐γ and IL‐17A production: BM ‐MNCs were cultured for 24 h. At the 0 h time‐point, whole cell lysate of Leishmania donovani antigen (10 µg/ml) and at the 18 h time‐point, monensin was supplemented in culture. Finally, at the end of 24 h of culture, cells were harvested and used for intracellular staining of IFN‐γ‐ and IL‐17A‐producing cells using anti‐human IFN‐γ and IL‐17A monoclonal antibodies along with anti‐human CD4 antibodies. (a) Scatter‐plot of IFN‐γ‐producing cells on gated CD4 + T cells: significantly decreased percentage frequency of IFN‐γ‐producing cells in patients with HPL ( n = 11: mean ± s.d.: 0·87 ± 0·13%) in comparison with patients with LPL ( n = 13: mean ± s.d.: 2·72 ± 0·54%) ( P = 0·005). (b) Scatter‐plot of IL‐17A‐producing cells on gated CD4 + T cells: as of IFN‐γ; the percentage frequency of IL‐17A also follows the same trend between HPL ( n = 11: mean ± s.d.: 0·68 ± 0·12%) and low parasitic load (LPL) ( n = 13: mean ± s.d.: 1·83 ± 0·29%) ( P

    Journal: Clinical and Experimental Immunology

    Article Title: T cell suppression in the bone marrow of visceral leishmaniasis patients: impact of parasite load

    doi: 10.1111/cei.13074

    Figure Lengend Snippet: Decreased frequency of interferon (IFN)‐γ‐ and interleukin (IL)‐17A‐producing cells in visceral leishmaniasis (VL) patients with high parasitic load (HPL). Enumeration of IFN‐γ‐ and IL‐17A‐producing cells in bone marrow mononuclear cells (BM MNCs): isolated MNCs ( n = 24) from bone marrow of VL patients were cultured for IFN‐γ and IL‐17A production: BM ‐MNCs were cultured for 24 h. At the 0 h time‐point, whole cell lysate of Leishmania donovani antigen (10 µg/ml) and at the 18 h time‐point, monensin was supplemented in culture. Finally, at the end of 24 h of culture, cells were harvested and used for intracellular staining of IFN‐γ‐ and IL‐17A‐producing cells using anti‐human IFN‐γ and IL‐17A monoclonal antibodies along with anti‐human CD4 antibodies. (a) Scatter‐plot of IFN‐γ‐producing cells on gated CD4 + T cells: significantly decreased percentage frequency of IFN‐γ‐producing cells in patients with HPL ( n = 11: mean ± s.d.: 0·87 ± 0·13%) in comparison with patients with LPL ( n = 13: mean ± s.d.: 2·72 ± 0·54%) ( P = 0·005). (b) Scatter‐plot of IL‐17A‐producing cells on gated CD4 + T cells: as of IFN‐γ; the percentage frequency of IL‐17A also follows the same trend between HPL ( n = 11: mean ± s.d.: 0·68 ± 0·12%) and low parasitic load (LPL) ( n = 13: mean ± s.d.: 1·83 ± 0·29%) ( P

    Article Snippet: Monensin (Golgi transport inhibitor; 1 mM; Sigma‐Aldrich) was added in the last 6 h of culture.

    Techniques: Isolation, Cell Culture, Staining

    Anti–TIM-4 inhibits IFN-γ expression by TIM-4 + B cells and augments IL-10 expression by TIM-1 + B cells in vitro. Purified B cells from IL-10–GFP reporter mice (BALB/c) were treated in vitro with LPS (2 μg/ml) and RIgG2a or anti–TIM-4 for 48 h. PMA, ionomycin, and monensin were added for the final 5 h. IL-10 expression was detected by GFP expression, whereas IFN-γ was detected by intracellular staining. Representative flow cytometry plots showing IFN-γ expression ( A ) or IL-10 expression ( C ) on CD19 + , CD19 + TIM-1 − TIM-4 − (DN), CD19 + TIM-1 + , and CD19 + TIM-4 + B cells. Frequency (mean + SD) of IFN-γ ( B ) or IL-10 ( D ) on cells in (A) ( n = 3). * p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: TIM-4 Identifies IFN-γ–Expressing Proinflammatory B Effector 1 Cells That Promote Tumor and Allograft Rejection

    doi: 10.4049/jimmunol.1602107

    Figure Lengend Snippet: Anti–TIM-4 inhibits IFN-γ expression by TIM-4 + B cells and augments IL-10 expression by TIM-1 + B cells in vitro. Purified B cells from IL-10–GFP reporter mice (BALB/c) were treated in vitro with LPS (2 μg/ml) and RIgG2a or anti–TIM-4 for 48 h. PMA, ionomycin, and monensin were added for the final 5 h. IL-10 expression was detected by GFP expression, whereas IFN-γ was detected by intracellular staining. Representative flow cytometry plots showing IFN-γ expression ( A ) or IL-10 expression ( C ) on CD19 + , CD19 + TIM-1 − TIM-4 − (DN), CD19 + TIM-1 + , and CD19 + TIM-4 + B cells. Frequency (mean + SD) of IFN-γ ( B ) or IL-10 ( D ) on cells in (A) ( n = 3). * p

    Article Snippet: The cells were plated at 1 × 106 /ml and stimulated with PMA (50 ng/ml), ionomycin (500 ng/ml), and monensin (1:1000) for 4–5 h (Sigma Aldrich) for phenotypic assessment of infiltrating lymphocytes and their intracellular cytokine expression.

    Techniques: Expressing, In Vitro, Purification, Mouse Assay, Staining, Flow Cytometry, Cytometry

    A Model Integrating Na V in Regeneration By 6 hpa, the H + pump V-ATPase is expressed in the regeneration bud where it regulates the membrane voltage of the bud. V-ATPase activation results in the up-regulation of Na V 1.2 by 18 hpa. Ablation of Na V 1.2 expression (RNAi) or Na V function (pharmacological treatment) inhibits regeneration. Na V activity enables sodium ions to enter regeneration bud cells and, potentially through SIK, to activate downstream pathways (such as BMP and Notch) by 24 hpa, driving regenerative outgrowth and patterning. By 7 days after injury, the rebuilding of the tail is largely complete. Importantly, monensin-mediated induction of a transient sodium flux into non-regenerative buds is sufficient to restore full tail regeneration, demonstrating that intracellular sodium signaling is a key regulator of regeneration able to initiate repair even after a non-regenerative wound epithelium has formed.

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Induction of Vertebrate Regeneration by a Transient Sodium Current

    doi: 10.1523/JNEUROSCI.3315-10.2010

    Figure Lengend Snippet: A Model Integrating Na V in Regeneration By 6 hpa, the H + pump V-ATPase is expressed in the regeneration bud where it regulates the membrane voltage of the bud. V-ATPase activation results in the up-regulation of Na V 1.2 by 18 hpa. Ablation of Na V 1.2 expression (RNAi) or Na V function (pharmacological treatment) inhibits regeneration. Na V activity enables sodium ions to enter regeneration bud cells and, potentially through SIK, to activate downstream pathways (such as BMP and Notch) by 24 hpa, driving regenerative outgrowth and patterning. By 7 days after injury, the rebuilding of the tail is largely complete. Importantly, monensin-mediated induction of a transient sodium flux into non-regenerative buds is sufficient to restore full tail regeneration, demonstrating that intracellular sodium signaling is a key regulator of regeneration able to initiate repair even after a non-regenerative wound epithelium has formed.

    Article Snippet: For refractory period analysis, tails were amputated at st. 46–47, and at 18 hpa animals were treated with or without 90mM sodium and 20µM monensin (Sigma) in 0.1X MMR with 90µM CoroNa Green for 45 min and washed twice in 0.1X MMR and 50µM BTS.

    Techniques: Activation Assay, Expressing, Activity Assay

    Transient Induction of Sodium Current Drives Regeneration ( A ) Regeneration rescue by human Na V 1.5. Control tail stumps (β-gal-injected) cut during the refractory period regenerate poorly, which is rescued by hNa V 1.5 expression. ( B ) Effects of regeneration rescue by hNa V 1.5 during refractory period block. ( C ) CoroNa Green analysis of Sodium current induction. ( C1 ) Control (vehicle only), non-induced refractory stage bud has little CoroNa Green signal. ( C2 ) Induction with 90mM sodium and 20µM monensin for 1 hour (18–19 hpa) significantly increases intracellular sodium (green). Images are merged brightfield and fluorescence of the same exposure time. white circle = refractory bud. ( C3 ) Most refractory stage amputations fail to regenerate. ( C4 ) Stimulation with sodium current restores full regeneration. ( D ) Transient sodium current rescues non-regenerative wound epidermis. Stimulation of sodium current increased regeneration > 2-fold and improved regeneration quality as compared to control siblings (treated with vehicle only: 0.01% ethanol). Treatment with either monensin or 90mM sodium alone showed no effect. Scale bars: ( A ) 1mm, ( C ) 500µm.

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Induction of Vertebrate Regeneration by a Transient Sodium Current

    doi: 10.1523/JNEUROSCI.3315-10.2010

    Figure Lengend Snippet: Transient Induction of Sodium Current Drives Regeneration ( A ) Regeneration rescue by human Na V 1.5. Control tail stumps (β-gal-injected) cut during the refractory period regenerate poorly, which is rescued by hNa V 1.5 expression. ( B ) Effects of regeneration rescue by hNa V 1.5 during refractory period block. ( C ) CoroNa Green analysis of Sodium current induction. ( C1 ) Control (vehicle only), non-induced refractory stage bud has little CoroNa Green signal. ( C2 ) Induction with 90mM sodium and 20µM monensin for 1 hour (18–19 hpa) significantly increases intracellular sodium (green). Images are merged brightfield and fluorescence of the same exposure time. white circle = refractory bud. ( C3 ) Most refractory stage amputations fail to regenerate. ( C4 ) Stimulation with sodium current restores full regeneration. ( D ) Transient sodium current rescues non-regenerative wound epidermis. Stimulation of sodium current increased regeneration > 2-fold and improved regeneration quality as compared to control siblings (treated with vehicle only: 0.01% ethanol). Treatment with either monensin or 90mM sodium alone showed no effect. Scale bars: ( A ) 1mm, ( C ) 500µm.

    Article Snippet: For refractory period analysis, tails were amputated at st. 46–47, and at 18 hpa animals were treated with or without 90mM sodium and 20µM monensin (Sigma) in 0.1X MMR with 90µM CoroNa Green for 45 min and washed twice in 0.1X MMR and 50µM BTS.

    Techniques: Injection, Expressing, Blocking Assay, Fluorescence