m egta buffered zero calcium dmem  (Thermo Fisher)


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    Structured Review

    Thermo Fisher m egta buffered zero calcium dmem
    M Egta Buffered Zero Calcium Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Article Title: Dendrites Contain a Spacing Pattern
    Article Snippet: Existing media were removed and replaced with 1 m m EGTA buffered zero-calcium DMEM (Invitrogen) for 2 min, which was in turn removed and replaced with more buffered DMEM for an additional 15 min.

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    Thermo Fisher alexa fluor 488 conjugate
    G-actin incorporation into filaments in dysferlin-deficient myoblasts. Fluorescently tagged G-actin incorporation into filaments was assayed in myoblasts permeabilized with 20 µM digitonin in the presence of 300 nM <t>Alexa-Fluor-488</t> actin, 2 mM ATP-Mg 2+ , and 10 µM free Ca 2+ during 6 min at 37 °C. After permeabilization, cells were fixed and nuclei were stained with DAPI. Confocal images were acquired at the equatorial plane of the cells using identical exposure settings between compared samples. ( a – c ) Fluorescent actin filaments in control C25 and dysferlinopathy DYSF2, DYSF3, AB320, and ER myoblasts ( a ) and RCMH myoblasts non-transfected (N-T) or stably transfected with shRNA for dysferlin ( c ). Scale bar = 20 µm. Insets show digital zooms of the boxed areas; brightness and contrast were increased to appreciate better the pattern of fluorescent actin. ( b – d ) Data are means ± SEM. Actin fluorescence intensity was measured in a single focal plane at the equator of cells and normalized by the cell area. The number of analyzed cells from five different cultures is indicated in parentheses. * p
    Alexa Fluor 488 Conjugate, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher gluoc
    MLKL phosphorylation and trimerization in <t>3T3-L1</t> adipocytes induced by high-dose <t>GluOC</t> in a manner dependent on FasL-Fas signaling. a Immunoblot analysis of total and phosphorylated (Thr 357 /Ser 358 ) forms of MLKL, as well as of RIP1 and RIP3 in 3T3-L1 adipocytes incubated with GluOC (5 or 40 ng/ml) for 3 or 24 h. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of total MLKL or of β-actin) from four independent experiments are shown. ** p
    Gluoc, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher anti p2x4
    P2X7 constitutes the low-affinity spermatogonial ATP sensor. (A) Exemplary whole-cell voltage-clamp recordings of currents triggered by repetitive stimulation (1 s; ISI = 3 s; S 1 , S 6 ) with ATP (300 µM; left) or BzATP (300 µM; right), respectively. Traces from untransfected spermatogonia (black) and cells transfected with either nontarget negative control siRNA (gray) or a construct targeting <t>P2X4</t> (green) are overlaid. (B) Quantification of results from repetitive stimulation experiments as shown in A. Peak current densities (means ± SEM) are color coded and plotted as a function of stimulus repetition. Cells were analyzed under control conditions ( n = 33/13 [ATP/BzATP]; black), after transfection with nontargeting siRNA ( n = 16/23 [ATP/BzATP]; gray), and after P2X4 knockdown ( n = 16/23 [ATP/BzATP]; green). Inset (left) shows the data delimited by the dashed rectangle at an increased scale. Asterisks (*) indicate statistical significance, P
    Anti P2x4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher flag tagged fxr
    Increased Akt activity and reduced FoxO3a binding in Map1lc3b promoter sites in acute ethanol-treated <t>FXR</t> KO mouse livers. Age matched WT and FXR KO mice were treated with 4.5 g/kg ethanol or water by gavage for 16 h. Total liver lysates were subjected to immunoblot analysis for serine 473 phosphorylated and total AKT, serine 9 phosphorylated and total GSK3β (A), and serine 253 phosphorylated and total FoxO3a (B). Densitometry analysis data are presented as a ratio of control ( n =3–4). Chromatin immunoprecipitation using FoxO3a antibody was performed to probe the three putative FoxO3a binding sites in Map1lc3b promoter site. DNA was isolated from chromatin pull-down and qPCR analysis was performed, and data was normalized to input chromatin. Relative binding abundance in comparison to untreated WT control was presented in (C). * p
    Flag Tagged Fxr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    G-actin incorporation into filaments in dysferlin-deficient myoblasts. Fluorescently tagged G-actin incorporation into filaments was assayed in myoblasts permeabilized with 20 µM digitonin in the presence of 300 nM Alexa-Fluor-488 actin, 2 mM ATP-Mg 2+ , and 10 µM free Ca 2+ during 6 min at 37 °C. After permeabilization, cells were fixed and nuclei were stained with DAPI. Confocal images were acquired at the equatorial plane of the cells using identical exposure settings between compared samples. ( a – c ) Fluorescent actin filaments in control C25 and dysferlinopathy DYSF2, DYSF3, AB320, and ER myoblasts ( a ) and RCMH myoblasts non-transfected (N-T) or stably transfected with shRNA for dysferlin ( c ). Scale bar = 20 µm. Insets show digital zooms of the boxed areas; brightness and contrast were increased to appreciate better the pattern of fluorescent actin. ( b – d ) Data are means ± SEM. Actin fluorescence intensity was measured in a single focal plane at the equator of cells and normalized by the cell area. The number of analyzed cells from five different cultures is indicated in parentheses. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Defects in G-Actin Incorporation into Filaments in Myoblasts Derived from Dysferlinopathy Patients Are Restored by Dysferlin C2 Domains

    doi: 10.3390/ijms21010037

    Figure Lengend Snippet: G-actin incorporation into filaments in dysferlin-deficient myoblasts. Fluorescently tagged G-actin incorporation into filaments was assayed in myoblasts permeabilized with 20 µM digitonin in the presence of 300 nM Alexa-Fluor-488 actin, 2 mM ATP-Mg 2+ , and 10 µM free Ca 2+ during 6 min at 37 °C. After permeabilization, cells were fixed and nuclei were stained with DAPI. Confocal images were acquired at the equatorial plane of the cells using identical exposure settings between compared samples. ( a – c ) Fluorescent actin filaments in control C25 and dysferlinopathy DYSF2, DYSF3, AB320, and ER myoblasts ( a ) and RCMH myoblasts non-transfected (N-T) or stably transfected with shRNA for dysferlin ( c ). Scale bar = 20 µm. Insets show digital zooms of the boxed areas; brightness and contrast were increased to appreciate better the pattern of fluorescent actin. ( b – d ) Data are means ± SEM. Actin fluorescence intensity was measured in a single focal plane at the equator of cells and normalized by the cell area. The number of analyzed cells from five different cultures is indicated in parentheses. * p

    Article Snippet: Reagents Actin, from rabbit muscle, Alexa Fluor™ 488 conjugate (Thermo Fisher Scientific, Waltham, MA, USA); ATP (Sigma-Aldrich, St. Louis, MO, USA); bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA); dexamethasone (Sigma-Aldrich, St. Louis, MO, USA); 40,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO, USA); digitonin (Sigma-Aldrich St. Louis, MO, USA); dimethyl sulfoxide (Merck Company, Darmstadt, Germany); DMEM/F-12 medium (Gibco, BRL, Gaithersburg, MD, USA); Dulbecco modified Eagle’s minimal essential medium (Gibco BRL, Gaithersburg, MD, USA); EDTA (Calbiochem, La Jolla, CA); EGTA (Sigma-Aldrich, St. Louis, MO, USA); fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA); fetuin (Sigma-Aldrich, St. Louis, MO, USA); gentamicin (Gibco/Life Technology, China); glutamic acid (Sigma-Aldrich, St. Louis, MO, USA); HEPES (Calbiochem, La Jolla, CA); human insulin (Eli Lilly and company, Indianapolis, USA); Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA); 199 medium (Sigma-Aldrich St. Louis, MO, USA); NaF (Merck Company, Darmstadt, Germany); Na3 VO4 (Merck Company, Darmstadt, Germany); Opti-Mem (Gibco BRL, Gaithersburg, MD, USA); penicillin (OPKO, Santiago, Chile); p-formaldehyde (Sigma-Aldrich St. Louis, MO, USA); phenylmethyl sulfonylflouride (Sigma-Aldrich, St. Louis, MO, USA); PIPES (Sigma-Aldrich, St. Louis, MO, USA); poly-l-lysine (Sigma-Aldrich, St. Louis, MO, USA); recombinant human basic fibroblast growth factor (Gibco BRL, Gaithersburg, MD, USA); protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA); recombinant human epidermal growth factor (Gibco BRL, Gaithersburg, MD, USA); triton X-100 (Merck Company, Darmstadt, Germany); trypsin- EDTA 0.25% (Sigma-Aldrich, St. Louis, MO, USA); and Tween-20 (Merck Company, Darmstadt, Germany) were purchased.

    Techniques: Staining, Transfection, Stable Transfection, shRNA, Fluorescence

    Annexin A2 distribution in dysferlinopathy myoblasts. Distribution of annexin A2 was analyzed by immunofluorescence using an anti-annexin A2 antibody and Cy3-conjugated anti-rabbit secondary antibody in C25 and DYSF3 myoblasts permeabilized with 20 µM digitonin in the presence of 300 nM Alexa-Fluor-488 actin, 2 mM ATP-Mg 2+ , and 10 µM free Ca 2+ . Confocal images were captured at the equatorial plane of the cells using identical exposure settings between compared samples; therefore, annexin A2 distribution was measured in a single focal plane. ( a ) C25 and DYSF3 myoblasts immunostained with annexin A2 (red) and fluorescent G-actin incorporated into filaments (green). Scale bar = 20 µm. Insets show digital magnification of the boxed areas. ( b , c ) Bars represent means ± SEM of the ratio of the mean fluorescence intensity of the cytosol/whole cell of annexin A2 immunostaining ( b ) and Pearson correlation coefficient for colocalization of annexin A2 with fluorescent actin filaments ( c ). The number of analyzed cells from four different cultures is indicated in parentheses. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Defects in G-Actin Incorporation into Filaments in Myoblasts Derived from Dysferlinopathy Patients Are Restored by Dysferlin C2 Domains

    doi: 10.3390/ijms21010037

    Figure Lengend Snippet: Annexin A2 distribution in dysferlinopathy myoblasts. Distribution of annexin A2 was analyzed by immunofluorescence using an anti-annexin A2 antibody and Cy3-conjugated anti-rabbit secondary antibody in C25 and DYSF3 myoblasts permeabilized with 20 µM digitonin in the presence of 300 nM Alexa-Fluor-488 actin, 2 mM ATP-Mg 2+ , and 10 µM free Ca 2+ . Confocal images were captured at the equatorial plane of the cells using identical exposure settings between compared samples; therefore, annexin A2 distribution was measured in a single focal plane. ( a ) C25 and DYSF3 myoblasts immunostained with annexin A2 (red) and fluorescent G-actin incorporated into filaments (green). Scale bar = 20 µm. Insets show digital magnification of the boxed areas. ( b , c ) Bars represent means ± SEM of the ratio of the mean fluorescence intensity of the cytosol/whole cell of annexin A2 immunostaining ( b ) and Pearson correlation coefficient for colocalization of annexin A2 with fluorescent actin filaments ( c ). The number of analyzed cells from four different cultures is indicated in parentheses. * p

    Article Snippet: Reagents Actin, from rabbit muscle, Alexa Fluor™ 488 conjugate (Thermo Fisher Scientific, Waltham, MA, USA); ATP (Sigma-Aldrich, St. Louis, MO, USA); bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA); dexamethasone (Sigma-Aldrich, St. Louis, MO, USA); 40,6-diamidino-2-phenylindole (Sigma-Aldrich, St. Louis, MO, USA); digitonin (Sigma-Aldrich St. Louis, MO, USA); dimethyl sulfoxide (Merck Company, Darmstadt, Germany); DMEM/F-12 medium (Gibco, BRL, Gaithersburg, MD, USA); Dulbecco modified Eagle’s minimal essential medium (Gibco BRL, Gaithersburg, MD, USA); EDTA (Calbiochem, La Jolla, CA); EGTA (Sigma-Aldrich, St. Louis, MO, USA); fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA); fetuin (Sigma-Aldrich, St. Louis, MO, USA); gentamicin (Gibco/Life Technology, China); glutamic acid (Sigma-Aldrich, St. Louis, MO, USA); HEPES (Calbiochem, La Jolla, CA); human insulin (Eli Lilly and company, Indianapolis, USA); Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA); 199 medium (Sigma-Aldrich St. Louis, MO, USA); NaF (Merck Company, Darmstadt, Germany); Na3 VO4 (Merck Company, Darmstadt, Germany); Opti-Mem (Gibco BRL, Gaithersburg, MD, USA); penicillin (OPKO, Santiago, Chile); p-formaldehyde (Sigma-Aldrich St. Louis, MO, USA); phenylmethyl sulfonylflouride (Sigma-Aldrich, St. Louis, MO, USA); PIPES (Sigma-Aldrich, St. Louis, MO, USA); poly-l-lysine (Sigma-Aldrich, St. Louis, MO, USA); recombinant human basic fibroblast growth factor (Gibco BRL, Gaithersburg, MD, USA); protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA); recombinant human epidermal growth factor (Gibco BRL, Gaithersburg, MD, USA); triton X-100 (Merck Company, Darmstadt, Germany); trypsin- EDTA 0.25% (Sigma-Aldrich, St. Louis, MO, USA); and Tween-20 (Merck Company, Darmstadt, Germany) were purchased.

    Techniques: Immunofluorescence, Fluorescence, Immunostaining

    MLKL phosphorylation and trimerization in 3T3-L1 adipocytes induced by high-dose GluOC in a manner dependent on FasL-Fas signaling. a Immunoblot analysis of total and phosphorylated (Thr 357 /Ser 358 ) forms of MLKL, as well as of RIP1 and RIP3 in 3T3-L1 adipocytes incubated with GluOC (5 or 40 ng/ml) for 3 or 24 h. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of total MLKL or of β-actin) from four independent experiments are shown. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: MLKL phosphorylation and trimerization in 3T3-L1 adipocytes induced by high-dose GluOC in a manner dependent on FasL-Fas signaling. a Immunoblot analysis of total and phosphorylated (Thr 357 /Ser 358 ) forms of MLKL, as well as of RIP1 and RIP3 in 3T3-L1 adipocytes incubated with GluOC (5 or 40 ng/ml) for 3 or 24 h. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of total MLKL or of β-actin) from four independent experiments are shown. ** p

    Article Snippet: Fluo-4 imaging of Ca2+ 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Incubation

    Calcium influx, as well as ROS and lipid peroxide production induced by high-dose GluOC in 3T3-L1 adipocytes. a Cells were incubated with GluOC (5 or 40 ng/ml) in the absence or presence of extracellular Ca 2+ for 24 h and then loaded with Fluo-4 for fluorescence imaging of intracellular Ca 2+ (upper panels). The fluorescence images are also shown merged with differential interference contrast images (lower panels). Data are representative of three independent experiments. Scale bar, 100 µm. b Cells were incubated with GluOC (5 or 40 ng/ml) or 1 mM H 2 O 2 for 12 h, after which nuclei were stained with Hoechst 33342 (green) and ROS were detected with CellROX Oxidative Stress Reagent (red). Representative images, as well as quantitative data (means + SEM) from three independent experiments are shown. Scale bar, 100 µm. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Calcium influx, as well as ROS and lipid peroxide production induced by high-dose GluOC in 3T3-L1 adipocytes. a Cells were incubated with GluOC (5 or 40 ng/ml) in the absence or presence of extracellular Ca 2+ for 24 h and then loaded with Fluo-4 for fluorescence imaging of intracellular Ca 2+ (upper panels). The fluorescence images are also shown merged with differential interference contrast images (lower panels). Data are representative of three independent experiments. Scale bar, 100 µm. b Cells were incubated with GluOC (5 or 40 ng/ml) or 1 mM H 2 O 2 for 12 h, after which nuclei were stained with Hoechst 33342 (green) and ROS were detected with CellROX Oxidative Stress Reagent (red). Representative images, as well as quantitative data (means + SEM) from three independent experiments are shown. Scale bar, 100 µm. ** p

    Article Snippet: Fluo-4 imaging of Ca2+ 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Incubation, Fluorescence, Imaging, Staining

    Activation of DRP1 and mitochondrial fragmentation induced by high-dose GluOC in 3T3-L1 adipocytes. a Cells were exposed to GluOC (5 or 40 ng/ml) for 3–24 h, after which cell lysates were subjected to immunoblot analysis with antibodies to total or Ser 637 - or Ser 616 -phosphorylated forms of DRP1. A representative blot, as well as quantitative data (means + SEM) from four independent experiments are shown. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Activation of DRP1 and mitochondrial fragmentation induced by high-dose GluOC in 3T3-L1 adipocytes. a Cells were exposed to GluOC (5 or 40 ng/ml) for 3–24 h, after which cell lysates were subjected to immunoblot analysis with antibodies to total or Ser 637 - or Ser 616 -phosphorylated forms of DRP1. A representative blot, as well as quantitative data (means + SEM) from four independent experiments are shown. ** p

    Article Snippet: Fluo-4 imaging of Ca2+ 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Activation Assay

    Effects of high-dose GluOC on the morphology and number of 3T3-L1 adipocytes. a Representative phase-contrast microscopic images of 3T3-L1 adipocytes or preadipocytes cultured with GluOC (5 or 40 ng/ml) or with 1 μM staurosporine (STS) for 48 or 96 h. Scale bars, 200 µm. b Cell counts determined from images as in a . Data are expressed as a percentage of the initial value and are means + SEM from three independent experiment. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Effects of high-dose GluOC on the morphology and number of 3T3-L1 adipocytes. a Representative phase-contrast microscopic images of 3T3-L1 adipocytes or preadipocytes cultured with GluOC (5 or 40 ng/ml) or with 1 μM staurosporine (STS) for 48 or 96 h. Scale bars, 200 µm. b Cell counts determined from images as in a . Data are expressed as a percentage of the initial value and are means + SEM from three independent experiment. ** p

    Article Snippet: Fluo-4 imaging of Ca2+ 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Cell Culture

    Role of GPRC6A signaling in high-dose GluOC action in 3T3-L1 adipocytes. a Cells were incubated with the indicated concentrations of GluOC or 1 μM staurosporine for 6 h, lysed, and subjected to immunoblot analysis of ATGL, total or Ser 522 -phosphorylated (p) forms of perilipin, CGI-58, FoxO1, FasL, adiponectin, and PPARγ. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of β-actin or, in the case of phospho-perilipin, by the amount of total perilipin) from three independent experiments are shown. * p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Role of GPRC6A signaling in high-dose GluOC action in 3T3-L1 adipocytes. a Cells were incubated with the indicated concentrations of GluOC or 1 μM staurosporine for 6 h, lysed, and subjected to immunoblot analysis of ATGL, total or Ser 522 -phosphorylated (p) forms of perilipin, CGI-58, FoxO1, FasL, adiponectin, and PPARγ. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of β-actin or, in the case of phospho-perilipin, by the amount of total perilipin) from three independent experiments are shown. * p

    Article Snippet: Fluo-4 imaging of Ca2+ 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Incubation

    Effects of high-dose GluOC on the expression and localization of FasL and consequent induction of necroptosis in 3T3-L1 adipocytes. a Immunoblot analysis of caspase-8 and caspase-3 (cleaved or full-length) in 3T3-L1 adipocytes or preadipocytes incubated with the indicated concentrations of GluOC or with 1 μM staurosporine for 6 h. The blot is representative of four independent experiments. b Fluorescence microscopic images of 3T3-L1 adipocytes exposed to the indicated concentrations of GluOC or 1 μM staurosporine for 48 h, or of 3T3-L1 preadipocytes exposed to 1 μM staurosporine for 1 h. The cells were stained with Hoechst 33342 (blue), EthD-III (red), and FITC-conjugated Annexin V (green). Scale bar, 200 µm. The images are representative of five independent experiments. c Immunoblot analysis of FasL and N-cadherin (loading control) in the plasma membrane (PM) fraction isolated from 3T3-L1 adipocytes after incubation with the indicated concentrations of GluOC for 12 h. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of N-cadherin) from three independent experiments are shown. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Effects of high-dose GluOC on the expression and localization of FasL and consequent induction of necroptosis in 3T3-L1 adipocytes. a Immunoblot analysis of caspase-8 and caspase-3 (cleaved or full-length) in 3T3-L1 adipocytes or preadipocytes incubated with the indicated concentrations of GluOC or with 1 μM staurosporine for 6 h. The blot is representative of four independent experiments. b Fluorescence microscopic images of 3T3-L1 adipocytes exposed to the indicated concentrations of GluOC or 1 μM staurosporine for 48 h, or of 3T3-L1 preadipocytes exposed to 1 μM staurosporine for 1 h. The cells were stained with Hoechst 33342 (blue), EthD-III (red), and FITC-conjugated Annexin V (green). Scale bar, 200 µm. The images are representative of five independent experiments. c Immunoblot analysis of FasL and N-cadherin (loading control) in the plasma membrane (PM) fraction isolated from 3T3-L1 adipocytes after incubation with the indicated concentrations of GluOC for 12 h. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of N-cadherin) from three independent experiments are shown. ** p

    Article Snippet: Fluo-4 imaging of Ca2+ 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Expressing, Incubation, Fluorescence, Staining, Ethidium Homodimer Assay, Isolation

    Role of CREB transcriptional co-activators in the upregulation of FoxO1 expression by high-dose GluOC in 3T3-L1 adipocytes. a Immunoblot analysis of cytoplasmic and nuclear fractions isolated from 3T3-L1 adipocytes after stimulation with GluOC (5 or 40 mg/ml) for 6 h. The blot is representative of five independent experiments. The relative amount of cytoplasmic and nuclear fractions analyzed was adjusted so as to obtain appropriate band intensities. Lamin B1 and α-tubulin were examined as loading controls for nuclear and cytoplasmic fractions, respectively. b HTRF analysis of the p300–CREB interaction in 3T3-L1 adipocytes incubated first in the absence or presence of 10 μM myristoylated PKI 14-22 amide or 10 µM U0126 and then in the additional absence or presence of GluOC (5 or 40 ng/ml) for 6 h. Data are means + SEM from 10 independent experiments. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Role of CREB transcriptional co-activators in the upregulation of FoxO1 expression by high-dose GluOC in 3T3-L1 adipocytes. a Immunoblot analysis of cytoplasmic and nuclear fractions isolated from 3T3-L1 adipocytes after stimulation with GluOC (5 or 40 mg/ml) for 6 h. The blot is representative of five independent experiments. The relative amount of cytoplasmic and nuclear fractions analyzed was adjusted so as to obtain appropriate band intensities. Lamin B1 and α-tubulin were examined as loading controls for nuclear and cytoplasmic fractions, respectively. b HTRF analysis of the p300–CREB interaction in 3T3-L1 adipocytes incubated first in the absence or presence of 10 μM myristoylated PKI 14-22 amide or 10 µM U0126 and then in the additional absence or presence of GluOC (5 or 40 ng/ml) for 6 h. Data are means + SEM from 10 independent experiments. ** p

    Article Snippet: Fluo-4 imaging of Ca2+ 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Expressing, Isolation, Incubation

    MLKL phosphorylation and trimerization in 3T3-L1 adipocytes induced by high-dose GluOC in a manner dependent on FasL-Fas signaling. a Immunoblot analysis of total and phosphorylated (Thr 357 /Ser 358 ) forms of MLKL, as well as of RIP1 and RIP3 in 3T3-L1 adipocytes incubated with GluOC (5 or 40 ng/ml) for 3 or 24 h. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of total MLKL or of β-actin) from four independent experiments are shown. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: MLKL phosphorylation and trimerization in 3T3-L1 adipocytes induced by high-dose GluOC in a manner dependent on FasL-Fas signaling. a Immunoblot analysis of total and phosphorylated (Thr 357 /Ser 358 ) forms of MLKL, as well as of RIP1 and RIP3 in 3T3-L1 adipocytes incubated with GluOC (5 or 40 ng/ml) for 3 or 24 h. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of total MLKL or of β-actin) from four independent experiments are shown. ** p

    Article Snippet: 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Incubation

    Calcium influx, as well as ROS and lipid peroxide production induced by high-dose GluOC in 3T3-L1 adipocytes. a Cells were incubated with GluOC (5 or 40 ng/ml) in the absence or presence of extracellular Ca 2+ for 24 h and then loaded with Fluo-4 for fluorescence imaging of intracellular Ca 2+ (upper panels). The fluorescence images are also shown merged with differential interference contrast images (lower panels). Data are representative of three independent experiments. Scale bar, 100 µm. b Cells were incubated with GluOC (5 or 40 ng/ml) or 1 mM H 2 O 2 for 12 h, after which nuclei were stained with Hoechst 33342 (green) and ROS were detected with CellROX Oxidative Stress Reagent (red). Representative images, as well as quantitative data (means + SEM) from three independent experiments are shown. Scale bar, 100 µm. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Calcium influx, as well as ROS and lipid peroxide production induced by high-dose GluOC in 3T3-L1 adipocytes. a Cells were incubated with GluOC (5 or 40 ng/ml) in the absence or presence of extracellular Ca 2+ for 24 h and then loaded with Fluo-4 for fluorescence imaging of intracellular Ca 2+ (upper panels). The fluorescence images are also shown merged with differential interference contrast images (lower panels). Data are representative of three independent experiments. Scale bar, 100 µm. b Cells were incubated with GluOC (5 or 40 ng/ml) or 1 mM H 2 O 2 for 12 h, after which nuclei were stained with Hoechst 33342 (green) and ROS were detected with CellROX Oxidative Stress Reagent (red). Representative images, as well as quantitative data (means + SEM) from three independent experiments are shown. Scale bar, 100 µm. ** p

    Article Snippet: 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Incubation, Fluorescence, Imaging, Staining

    Activation of DRP1 and mitochondrial fragmentation induced by high-dose GluOC in 3T3-L1 adipocytes. a Cells were exposed to GluOC (5 or 40 ng/ml) for 3–24 h, after which cell lysates were subjected to immunoblot analysis with antibodies to total or Ser 637 - or Ser 616 -phosphorylated forms of DRP1. A representative blot, as well as quantitative data (means + SEM) from four independent experiments are shown. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Activation of DRP1 and mitochondrial fragmentation induced by high-dose GluOC in 3T3-L1 adipocytes. a Cells were exposed to GluOC (5 or 40 ng/ml) for 3–24 h, after which cell lysates were subjected to immunoblot analysis with antibodies to total or Ser 637 - or Ser 616 -phosphorylated forms of DRP1. A representative blot, as well as quantitative data (means + SEM) from four independent experiments are shown. ** p

    Article Snippet: 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Activation Assay

    Effects of high-dose GluOC on the morphology and number of 3T3-L1 adipocytes. a Representative phase-contrast microscopic images of 3T3-L1 adipocytes or preadipocytes cultured with GluOC (5 or 40 ng/ml) or with 1 μM staurosporine (STS) for 48 or 96 h. Scale bars, 200 µm. b Cell counts determined from images as in a . Data are expressed as a percentage of the initial value and are means + SEM from three independent experiment. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Effects of high-dose GluOC on the morphology and number of 3T3-L1 adipocytes. a Representative phase-contrast microscopic images of 3T3-L1 adipocytes or preadipocytes cultured with GluOC (5 or 40 ng/ml) or with 1 μM staurosporine (STS) for 48 or 96 h. Scale bars, 200 µm. b Cell counts determined from images as in a . Data are expressed as a percentage of the initial value and are means + SEM from three independent experiment. ** p

    Article Snippet: 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Cell Culture

    Role of GPRC6A signaling in high-dose GluOC action in 3T3-L1 adipocytes. a Cells were incubated with the indicated concentrations of GluOC or 1 μM staurosporine for 6 h, lysed, and subjected to immunoblot analysis of ATGL, total or Ser 522 -phosphorylated (p) forms of perilipin, CGI-58, FoxO1, FasL, adiponectin, and PPARγ. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of β-actin or, in the case of phospho-perilipin, by the amount of total perilipin) from three independent experiments are shown. * p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Role of GPRC6A signaling in high-dose GluOC action in 3T3-L1 adipocytes. a Cells were incubated with the indicated concentrations of GluOC or 1 μM staurosporine for 6 h, lysed, and subjected to immunoblot analysis of ATGL, total or Ser 522 -phosphorylated (p) forms of perilipin, CGI-58, FoxO1, FasL, adiponectin, and PPARγ. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of β-actin or, in the case of phospho-perilipin, by the amount of total perilipin) from three independent experiments are shown. * p

    Article Snippet: 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Incubation

    Effects of high-dose GluOC on the expression and localization of FasL and consequent induction of necroptosis in 3T3-L1 adipocytes. a Immunoblot analysis of caspase-8 and caspase-3 (cleaved or full-length) in 3T3-L1 adipocytes or preadipocytes incubated with the indicated concentrations of GluOC or with 1 μM staurosporine for 6 h. The blot is representative of four independent experiments. b Fluorescence microscopic images of 3T3-L1 adipocytes exposed to the indicated concentrations of GluOC or 1 μM staurosporine for 48 h, or of 3T3-L1 preadipocytes exposed to 1 μM staurosporine for 1 h. The cells were stained with Hoechst 33342 (blue), EthD-III (red), and FITC-conjugated Annexin V (green). Scale bar, 200 µm. The images are representative of five independent experiments. c Immunoblot analysis of FasL and N-cadherin (loading control) in the plasma membrane (PM) fraction isolated from 3T3-L1 adipocytes after incubation with the indicated concentrations of GluOC for 12 h. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of N-cadherin) from three independent experiments are shown. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Effects of high-dose GluOC on the expression and localization of FasL and consequent induction of necroptosis in 3T3-L1 adipocytes. a Immunoblot analysis of caspase-8 and caspase-3 (cleaved or full-length) in 3T3-L1 adipocytes or preadipocytes incubated with the indicated concentrations of GluOC or with 1 μM staurosporine for 6 h. The blot is representative of four independent experiments. b Fluorescence microscopic images of 3T3-L1 adipocytes exposed to the indicated concentrations of GluOC or 1 μM staurosporine for 48 h, or of 3T3-L1 preadipocytes exposed to 1 μM staurosporine for 1 h. The cells were stained with Hoechst 33342 (blue), EthD-III (red), and FITC-conjugated Annexin V (green). Scale bar, 200 µm. The images are representative of five independent experiments. c Immunoblot analysis of FasL and N-cadherin (loading control) in the plasma membrane (PM) fraction isolated from 3T3-L1 adipocytes after incubation with the indicated concentrations of GluOC for 12 h. A representative blot, as well as quantitative data (means + SEM, normalized by the amount of N-cadherin) from three independent experiments are shown. ** p

    Article Snippet: 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Expressing, Incubation, Fluorescence, Staining, Ethidium Homodimer Assay, Isolation

    Role of CREB transcriptional co-activators in the upregulation of FoxO1 expression by high-dose GluOC in 3T3-L1 adipocytes. a Immunoblot analysis of cytoplasmic and nuclear fractions isolated from 3T3-L1 adipocytes after stimulation with GluOC (5 or 40 mg/ml) for 6 h. The blot is representative of five independent experiments. The relative amount of cytoplasmic and nuclear fractions analyzed was adjusted so as to obtain appropriate band intensities. Lamin B1 and α-tubulin were examined as loading controls for nuclear and cytoplasmic fractions, respectively. b HTRF analysis of the p300–CREB interaction in 3T3-L1 adipocytes incubated first in the absence or presence of 10 μM myristoylated PKI 14-22 amide or 10 µM U0126 and then in the additional absence or presence of GluOC (5 or 40 ng/ml) for 6 h. Data are means + SEM from 10 independent experiments. ** p

    Journal: Cell Death & Disease

    Article Title: Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300

    doi: 10.1038/s41419-018-1257-7

    Figure Lengend Snippet: Role of CREB transcriptional co-activators in the upregulation of FoxO1 expression by high-dose GluOC in 3T3-L1 adipocytes. a Immunoblot analysis of cytoplasmic and nuclear fractions isolated from 3T3-L1 adipocytes after stimulation with GluOC (5 or 40 mg/ml) for 6 h. The blot is representative of five independent experiments. The relative amount of cytoplasmic and nuclear fractions analyzed was adjusted so as to obtain appropriate band intensities. Lamin B1 and α-tubulin were examined as loading controls for nuclear and cytoplasmic fractions, respectively. b HTRF analysis of the p300–CREB interaction in 3T3-L1 adipocytes incubated first in the absence or presence of 10 μM myristoylated PKI 14-22 amide or 10 µM U0126 and then in the additional absence or presence of GluOC (5 or 40 ng/ml) for 6 h. Data are means + SEM from 10 independent experiments. ** p

    Article Snippet: 3T3-L1 adipocytes were exposed for 24 h to the indicated concentrations of GluOC either in DMEM or in Ca2+ -free DMEM containing 1 mM EGTA (Thermo Fisher Scientific).

    Techniques: Expressing, Isolation, Incubation

    P2X7 constitutes the low-affinity spermatogonial ATP sensor. (A) Exemplary whole-cell voltage-clamp recordings of currents triggered by repetitive stimulation (1 s; ISI = 3 s; S 1 , S 6 ) with ATP (300 µM; left) or BzATP (300 µM; right), respectively. Traces from untransfected spermatogonia (black) and cells transfected with either nontarget negative control siRNA (gray) or a construct targeting P2X4 (green) are overlaid. (B) Quantification of results from repetitive stimulation experiments as shown in A. Peak current densities (means ± SEM) are color coded and plotted as a function of stimulus repetition. Cells were analyzed under control conditions ( n = 33/13 [ATP/BzATP]; black), after transfection with nontargeting siRNA ( n = 16/23 [ATP/BzATP]; gray), and after P2X4 knockdown ( n = 16/23 [ATP/BzATP]; green). Inset (left) shows the data delimited by the dashed rectangle at an increased scale. Asterisks (*) indicate statistical significance, P

    Journal: The Journal of General Physiology

    Article Title: Distinct purinergic signaling pathways in prepubescent mouse spermatogonia

    doi: 10.1085/jgp.201611636

    Figure Lengend Snippet: P2X7 constitutes the low-affinity spermatogonial ATP sensor. (A) Exemplary whole-cell voltage-clamp recordings of currents triggered by repetitive stimulation (1 s; ISI = 3 s; S 1 , S 6 ) with ATP (300 µM; left) or BzATP (300 µM; right), respectively. Traces from untransfected spermatogonia (black) and cells transfected with either nontarget negative control siRNA (gray) or a construct targeting P2X4 (green) are overlaid. (B) Quantification of results from repetitive stimulation experiments as shown in A. Peak current densities (means ± SEM) are color coded and plotted as a function of stimulus repetition. Cells were analyzed under control conditions ( n = 33/13 [ATP/BzATP]; black), after transfection with nontargeting siRNA ( n = 16/23 [ATP/BzATP]; gray), and after P2X4 knockdown ( n = 16/23 [ATP/BzATP]; green). Inset (left) shows the data delimited by the dashed rectangle at an increased scale. Asterisks (*) indicate statistical significance, P

    Article Snippet: Chemicals and solutions The following solutions were used: (S1 ) HEPES-buffered extracellular solution containing (mM) 145 NaCl, 5 KCl, 1 CaCl2 , 0.5 MgCl2 , and 10 HEPES; pH = 7.3 (adjusted with NaOH); osmolarity = 300 mOsm (adjusted with glucose). (S2 ) Oxygenated (95% O2 , 5% CO2 ) extracellular solution containing (mM) 120 NaCl, 25 NaHCO3 , 5 KCl, 0.5 MgCl2 , 1.0 CaCl2 , 5 N ,N -bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), and 10 glucose; pH = 7.3 (NaOH); 300 mOsm (glucose). (S3 ) Extracellular low Ca2+ solution containing (mM) 145 NaCl, 5 KCl, 2.5 CaCl2 , 0.5 MgCl2 , 10 HEPES, and 5 EGTA; pH = 7.3 (NaOH); osmolarity = 300 mOsm (glucose), [Ca2+ ]free = ∼110 nM. (S4 ) Extracellular TEA solution containing (mM) 120 NaCl, 15 TEACl, 5 KCl, 1 CaCl2 , 0.5 MgCl2 , and 10 HEPES; pH = 7.3 (NaOH); osmolarity = 300 mOsm (glucose). (S5 ) Standard pipette solution containing (mM) 143 KCl, 2 KOH, 1 EGTA, 0.3 CaCl2 , 10 HEPES, and 1 Na-GTP ([Ca2+ ]free = 110 nM); pH = 7.1 (adjusted with KOH); osmolarity = 290 mOsm (glucose). (S6 ) Cs+ -based pipette solution containing (mM) 143 CsCl, 2 CsOH, 1 EGTA, 0.3 CaCl2 , 10 HEPES, and 1 Na-GTP ([Ca2+ ]free = 110 nM); pH = 7.1 (adjusted with CsOH); osmolarity = 290 mOsm (glucose). (S7 ) Standard blocking solution containing 5% (anti-P2X4, anti-P2X7, anti-Sloα1) or 10% (anti-DAZL) normal goat serum (Thermo Fisher Scientific), 10 mg/ml BSA, 0.3% Triton X-100, and 0.02% NaN3 in PBS−/− (100 mM). (S8 ) Cell culture blocking solution containing 1% BSA and 0.1% Triton X-100 in PBS−/− (100 mM). (S9 ) Standard washing solution containing 10 mg/ml BSA in PBS−/− (100 mM). (S10 ) Standard staining solution containing 3% BSA (IgG free, protease free), 0.05% NaN3 , and Alexa Fluor 488 or 633 streptavidin conjugate (1:800; Thermo Fisher Scientific) in PBS−/− (100 mM). (S11 ) Standard co-culture medium (adopted and modified from ) consisting of DMEM (low glucose, pyruvate; Thermo Fisher Scientific) complemented with 10 ng/ml epidermal growth factor, 10% fetal calf serum (Thermo Fisher Scientific), 1 ng/ml FSH, 0.2 ng/ml growth hormone releasing factor, 5 µg/ml insulin, 10 ng/ml insulin-like growth factor, 1% MEM nonessential amino acid solution (Thermo Fisher Scientific), 0.01% nucleoside solution, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µM retinol acetate, 0.5 mM sodium pyruvate, 100 nM testosterone, and 5 µg/ml transferrin.

    Techniques: Transfection, Negative Control, Construct

    Posttranscriptional gene silencing confirms P2X4 as the high-affinity spermatogonial ATP sensor. (A) Bar chart quantifying siRNA-dependent selective knockdown of spermatogonial gene expression in vitro. When transfected with one of two siRNA constructs (green), knockdown of P2X4 expression was confirmed by quantitative real-time PCR. Transcript quantities (means ± SEM) are normalized to mRNA levels in untransfected spermatogonia and compared with cells treated with nontargeting siRNA controls (gray). Administration of both targeting siRNAs effectively reduced P2X4 transcription as compared with both untransfected spermatogonia and negative (nontargeting siRNA) controls. Asterisks (*) denote statistical significance, P

    Journal: The Journal of General Physiology

    Article Title: Distinct purinergic signaling pathways in prepubescent mouse spermatogonia

    doi: 10.1085/jgp.201611636

    Figure Lengend Snippet: Posttranscriptional gene silencing confirms P2X4 as the high-affinity spermatogonial ATP sensor. (A) Bar chart quantifying siRNA-dependent selective knockdown of spermatogonial gene expression in vitro. When transfected with one of two siRNA constructs (green), knockdown of P2X4 expression was confirmed by quantitative real-time PCR. Transcript quantities (means ± SEM) are normalized to mRNA levels in untransfected spermatogonia and compared with cells treated with nontargeting siRNA controls (gray). Administration of both targeting siRNAs effectively reduced P2X4 transcription as compared with both untransfected spermatogonia and negative (nontargeting siRNA) controls. Asterisks (*) denote statistical significance, P

    Article Snippet: Chemicals and solutions The following solutions were used: (S1 ) HEPES-buffered extracellular solution containing (mM) 145 NaCl, 5 KCl, 1 CaCl2 , 0.5 MgCl2 , and 10 HEPES; pH = 7.3 (adjusted with NaOH); osmolarity = 300 mOsm (adjusted with glucose). (S2 ) Oxygenated (95% O2 , 5% CO2 ) extracellular solution containing (mM) 120 NaCl, 25 NaHCO3 , 5 KCl, 0.5 MgCl2 , 1.0 CaCl2 , 5 N ,N -bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), and 10 glucose; pH = 7.3 (NaOH); 300 mOsm (glucose). (S3 ) Extracellular low Ca2+ solution containing (mM) 145 NaCl, 5 KCl, 2.5 CaCl2 , 0.5 MgCl2 , 10 HEPES, and 5 EGTA; pH = 7.3 (NaOH); osmolarity = 300 mOsm (glucose), [Ca2+ ]free = ∼110 nM. (S4 ) Extracellular TEA solution containing (mM) 120 NaCl, 15 TEACl, 5 KCl, 1 CaCl2 , 0.5 MgCl2 , and 10 HEPES; pH = 7.3 (NaOH); osmolarity = 300 mOsm (glucose). (S5 ) Standard pipette solution containing (mM) 143 KCl, 2 KOH, 1 EGTA, 0.3 CaCl2 , 10 HEPES, and 1 Na-GTP ([Ca2+ ]free = 110 nM); pH = 7.1 (adjusted with KOH); osmolarity = 290 mOsm (glucose). (S6 ) Cs+ -based pipette solution containing (mM) 143 CsCl, 2 CsOH, 1 EGTA, 0.3 CaCl2 , 10 HEPES, and 1 Na-GTP ([Ca2+ ]free = 110 nM); pH = 7.1 (adjusted with CsOH); osmolarity = 290 mOsm (glucose). (S7 ) Standard blocking solution containing 5% (anti-P2X4, anti-P2X7, anti-Sloα1) or 10% (anti-DAZL) normal goat serum (Thermo Fisher Scientific), 10 mg/ml BSA, 0.3% Triton X-100, and 0.02% NaN3 in PBS−/− (100 mM). (S8 ) Cell culture blocking solution containing 1% BSA and 0.1% Triton X-100 in PBS−/− (100 mM). (S9 ) Standard washing solution containing 10 mg/ml BSA in PBS−/− (100 mM). (S10 ) Standard staining solution containing 3% BSA (IgG free, protease free), 0.05% NaN3 , and Alexa Fluor 488 or 633 streptavidin conjugate (1:800; Thermo Fisher Scientific) in PBS−/− (100 mM). (S11 ) Standard co-culture medium (adopted and modified from ) consisting of DMEM (low glucose, pyruvate; Thermo Fisher Scientific) complemented with 10 ng/ml epidermal growth factor, 10% fetal calf serum (Thermo Fisher Scientific), 1 ng/ml FSH, 0.2 ng/ml growth hormone releasing factor, 5 µg/ml insulin, 10 ng/ml insulin-like growth factor, 1% MEM nonessential amino acid solution (Thermo Fisher Scientific), 0.01% nucleoside solution, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µM retinol acetate, 0.5 mM sodium pyruvate, 100 nM testosterone, and 5 µg/ml transferrin.

    Techniques: Expressing, In Vitro, Transfection, Construct, Real-time Polymerase Chain Reaction

    P2X4 is a candidate mediator of the high-affinity ATP response in spermatogonia. (A) Reverse transcription PCR reveals transcripts for P2rx2 , P2rx4 , and P2rx7 in both Sertoli–germ cell co-cultures and whole testis preparations at P7. A faint band for P2rx3 -encoding cDNA is only amplified from whole testis samples but not co-cultures. Primer pairs generate amplificates of 150–250 bp, respectively. cDNA from whole brain/spinal cord serves as positive control, whereas no template controls are devoid of detectable signals. (B–E) Exemplary whole-cell voltage-clamp recordings of ATP-induced currents (10 µM; 1 s; V hold = −80 mV; S 1 , S 6 ) under control conditions and during treatment with suramin (10 µM and 100 µM; B), extracellular Cu 2+ (100 µM; C), reduced pH (6.3; D), and ivermectin (3 µM; E). Altered conditions were established for at least 60 s (preincubation). (F) Bar diagram (means ± SEM) depicting the quantitative analysis of experiments exemplified in B–E. Note the discontinuous ordinate (//). Numbers of cells tested are indicated above bars. Asterisks (*) denote statistical significance, P ≤ 0.01 (paired t test).

    Journal: The Journal of General Physiology

    Article Title: Distinct purinergic signaling pathways in prepubescent mouse spermatogonia

    doi: 10.1085/jgp.201611636

    Figure Lengend Snippet: P2X4 is a candidate mediator of the high-affinity ATP response in spermatogonia. (A) Reverse transcription PCR reveals transcripts for P2rx2 , P2rx4 , and P2rx7 in both Sertoli–germ cell co-cultures and whole testis preparations at P7. A faint band for P2rx3 -encoding cDNA is only amplified from whole testis samples but not co-cultures. Primer pairs generate amplificates of 150–250 bp, respectively. cDNA from whole brain/spinal cord serves as positive control, whereas no template controls are devoid of detectable signals. (B–E) Exemplary whole-cell voltage-clamp recordings of ATP-induced currents (10 µM; 1 s; V hold = −80 mV; S 1 , S 6 ) under control conditions and during treatment with suramin (10 µM and 100 µM; B), extracellular Cu 2+ (100 µM; C), reduced pH (6.3; D), and ivermectin (3 µM; E). Altered conditions were established for at least 60 s (preincubation). (F) Bar diagram (means ± SEM) depicting the quantitative analysis of experiments exemplified in B–E. Note the discontinuous ordinate (//). Numbers of cells tested are indicated above bars. Asterisks (*) denote statistical significance, P ≤ 0.01 (paired t test).

    Article Snippet: Chemicals and solutions The following solutions were used: (S1 ) HEPES-buffered extracellular solution containing (mM) 145 NaCl, 5 KCl, 1 CaCl2 , 0.5 MgCl2 , and 10 HEPES; pH = 7.3 (adjusted with NaOH); osmolarity = 300 mOsm (adjusted with glucose). (S2 ) Oxygenated (95% O2 , 5% CO2 ) extracellular solution containing (mM) 120 NaCl, 25 NaHCO3 , 5 KCl, 0.5 MgCl2 , 1.0 CaCl2 , 5 N ,N -bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), and 10 glucose; pH = 7.3 (NaOH); 300 mOsm (glucose). (S3 ) Extracellular low Ca2+ solution containing (mM) 145 NaCl, 5 KCl, 2.5 CaCl2 , 0.5 MgCl2 , 10 HEPES, and 5 EGTA; pH = 7.3 (NaOH); osmolarity = 300 mOsm (glucose), [Ca2+ ]free = ∼110 nM. (S4 ) Extracellular TEA solution containing (mM) 120 NaCl, 15 TEACl, 5 KCl, 1 CaCl2 , 0.5 MgCl2 , and 10 HEPES; pH = 7.3 (NaOH); osmolarity = 300 mOsm (glucose). (S5 ) Standard pipette solution containing (mM) 143 KCl, 2 KOH, 1 EGTA, 0.3 CaCl2 , 10 HEPES, and 1 Na-GTP ([Ca2+ ]free = 110 nM); pH = 7.1 (adjusted with KOH); osmolarity = 290 mOsm (glucose). (S6 ) Cs+ -based pipette solution containing (mM) 143 CsCl, 2 CsOH, 1 EGTA, 0.3 CaCl2 , 10 HEPES, and 1 Na-GTP ([Ca2+ ]free = 110 nM); pH = 7.1 (adjusted with CsOH); osmolarity = 290 mOsm (glucose). (S7 ) Standard blocking solution containing 5% (anti-P2X4, anti-P2X7, anti-Sloα1) or 10% (anti-DAZL) normal goat serum (Thermo Fisher Scientific), 10 mg/ml BSA, 0.3% Triton X-100, and 0.02% NaN3 in PBS−/− (100 mM). (S8 ) Cell culture blocking solution containing 1% BSA and 0.1% Triton X-100 in PBS−/− (100 mM). (S9 ) Standard washing solution containing 10 mg/ml BSA in PBS−/− (100 mM). (S10 ) Standard staining solution containing 3% BSA (IgG free, protease free), 0.05% NaN3 , and Alexa Fluor 488 or 633 streptavidin conjugate (1:800; Thermo Fisher Scientific) in PBS−/− (100 mM). (S11 ) Standard co-culture medium (adopted and modified from ) consisting of DMEM (low glucose, pyruvate; Thermo Fisher Scientific) complemented with 10 ng/ml epidermal growth factor, 10% fetal calf serum (Thermo Fisher Scientific), 1 ng/ml FSH, 0.2 ng/ml growth hormone releasing factor, 5 µg/ml insulin, 10 ng/ml insulin-like growth factor, 1% MEM nonessential amino acid solution (Thermo Fisher Scientific), 0.01% nucleoside solution, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 µM retinol acetate, 0.5 mM sodium pyruvate, 100 nM testosterone, and 5 µg/ml transferrin.

    Techniques: Polymerase Chain Reaction, Amplification, Positive Control

    Increased Akt activity and reduced FoxO3a binding in Map1lc3b promoter sites in acute ethanol-treated FXR KO mouse livers. Age matched WT and FXR KO mice were treated with 4.5 g/kg ethanol or water by gavage for 16 h. Total liver lysates were subjected to immunoblot analysis for serine 473 phosphorylated and total AKT, serine 9 phosphorylated and total GSK3β (A), and serine 253 phosphorylated and total FoxO3a (B). Densitometry analysis data are presented as a ratio of control ( n =3–4). Chromatin immunoprecipitation using FoxO3a antibody was performed to probe the three putative FoxO3a binding sites in Map1lc3b promoter site. DNA was isolated from chromatin pull-down and qPCR analysis was performed, and data was normalized to input chromatin. Relative binding abundance in comparison to untreated WT control was presented in (C). * p

    Journal: Redox Biology

    Article Title: Farnesoid X receptor regulates forkhead Box O3a activation in ethanol-induced autophagy and hepatotoxicity

    doi: 10.1016/j.redox.2014.08.007

    Figure Lengend Snippet: Increased Akt activity and reduced FoxO3a binding in Map1lc3b promoter sites in acute ethanol-treated FXR KO mouse livers. Age matched WT and FXR KO mice were treated with 4.5 g/kg ethanol or water by gavage for 16 h. Total liver lysates were subjected to immunoblot analysis for serine 473 phosphorylated and total AKT, serine 9 phosphorylated and total GSK3β (A), and serine 253 phosphorylated and total FoxO3a (B). Densitometry analysis data are presented as a ratio of control ( n =3–4). Chromatin immunoprecipitation using FoxO3a antibody was performed to probe the three putative FoxO3a binding sites in Map1lc3b promoter site. DNA was isolated from chromatin pull-down and qPCR analysis was performed, and data was normalized to input chromatin. Relative binding abundance in comparison to untreated WT control was presented in (C). * p

    Article Snippet: Cell culture and transfection Human embryonic kidney 293A cells were cultured in DMEM medium with FBS and l -glutamate and transiently transfected with HA tagged FoxO3, Flag tagged FXR, HA or Flag plasmid constructs using TurboFect Transfection reagent (Thermo, Fisher Scientific) for 24 h. Total cell lysates then were extracted using HA cell lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA pH 7.5, 1 mM EGTA pH 7.5, 1% Triton X-100, protease inhibitors).

    Techniques: Activity Assay, Binding Assay, Mouse Assay, Chromatin Immunoprecipitation, Isolation, Real-time Polymerase Chain Reaction

    Ethanol-induced FoxO3a nuclear translocation was diminished in FXR KO mouse livers. Age matched WT and FXR KO mice were treated with 4.5 g/kg ethanol or water by gavage for 16 h. Liver cyrosections were immunostained for FoxO3a and Hoechst 33342 for nuclei, and representative images are shown in (A). Nuclei positive for FoxO3a were quantified from at least 3 images (B). Cytosolic and nuclear fractions were isolated from liver and subjected to immunoblot analysis for FoxO3a and FXR (C) densitometry analysis data are presented as a ratio of control ( n =3).

    Journal: Redox Biology

    Article Title: Farnesoid X receptor regulates forkhead Box O3a activation in ethanol-induced autophagy and hepatotoxicity

    doi: 10.1016/j.redox.2014.08.007

    Figure Lengend Snippet: Ethanol-induced FoxO3a nuclear translocation was diminished in FXR KO mouse livers. Age matched WT and FXR KO mice were treated with 4.5 g/kg ethanol or water by gavage for 16 h. Liver cyrosections were immunostained for FoxO3a and Hoechst 33342 for nuclei, and representative images are shown in (A). Nuclei positive for FoxO3a were quantified from at least 3 images (B). Cytosolic and nuclear fractions were isolated from liver and subjected to immunoblot analysis for FoxO3a and FXR (C) densitometry analysis data are presented as a ratio of control ( n =3).

    Article Snippet: Cell culture and transfection Human embryonic kidney 293A cells were cultured in DMEM medium with FBS and l -glutamate and transiently transfected with HA tagged FoxO3, Flag tagged FXR, HA or Flag plasmid constructs using TurboFect Transfection reagent (Thermo, Fisher Scientific) for 24 h. Total cell lysates then were extracted using HA cell lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA pH 7.5, 1 mM EGTA pH 7.5, 1% Triton X-100, protease inhibitors).

    Techniques: Translocation Assay, Mouse Assay, Isolation

    Ethanol-induced FoxO3a-mediated transcription of autophagy related and FoxO3a target genes were inhibited in FXR KO mouse livers. Age matched WT and FXR KO mice were treated with 4.5 g/kg ethanol or water by gavage for 16 h. Hepatic mRNA was isolated and qRT-PCR was performed for autophagy related genes, Atg5 , Becn-1 , and MAP1LC3B (A) and FoxO3a target genes, MnSOD , p21 , and FoxO3a (B). qRT-PCR was also performed for FXR target gene, Shp (C). The gene expression levels were normalized to β-actin and shown as fold increase over wild type mice ( n =4–7). * p

    Journal: Redox Biology

    Article Title: Farnesoid X receptor regulates forkhead Box O3a activation in ethanol-induced autophagy and hepatotoxicity

    doi: 10.1016/j.redox.2014.08.007

    Figure Lengend Snippet: Ethanol-induced FoxO3a-mediated transcription of autophagy related and FoxO3a target genes were inhibited in FXR KO mouse livers. Age matched WT and FXR KO mice were treated with 4.5 g/kg ethanol or water by gavage for 16 h. Hepatic mRNA was isolated and qRT-PCR was performed for autophagy related genes, Atg5 , Becn-1 , and MAP1LC3B (A) and FoxO3a target genes, MnSOD , p21 , and FoxO3a (B). qRT-PCR was also performed for FXR target gene, Shp (C). The gene expression levels were normalized to β-actin and shown as fold increase over wild type mice ( n =4–7). * p

    Article Snippet: Cell culture and transfection Human embryonic kidney 293A cells were cultured in DMEM medium with FBS and l -glutamate and transiently transfected with HA tagged FoxO3, Flag tagged FXR, HA or Flag plasmid constructs using TurboFect Transfection reagent (Thermo, Fisher Scientific) for 24 h. Total cell lysates then were extracted using HA cell lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA pH 7.5, 1 mM EGTA pH 7.5, 1% Triton X-100, protease inhibitors).

    Techniques: Mouse Assay, Isolation, Quantitative RT-PCR, Expressing

    FXR and FoxO3a did not interact in vitro or in vivo. HEK 293A cells were transfected with plasmids containing flag-FXR, HA-FoxO3a, HA or flag. Total cell lysates were isolated, and flag was pulled down by immunoprecipitation and subjected to immunoblot analysis for flag and HA. Input total lysates from transfected HEK 293A were used as positive controls for FoxO3a and FXR (A). Cytosol and nuclear fractions were obtained from transfected HEK 293A cells, and immunoblot analysis was performed for flag and FoxO3 (B). Age matched WT and FXR KO mice were treated with 4.5 g/kg ethanol or water by gavage for 16 h. FoxO3a in total liver lysates was pulled down by immunopreciptation and subjected to immunoblot analysis for FXR and FoxO3a (C).

    Journal: Redox Biology

    Article Title: Farnesoid X receptor regulates forkhead Box O3a activation in ethanol-induced autophagy and hepatotoxicity

    doi: 10.1016/j.redox.2014.08.007

    Figure Lengend Snippet: FXR and FoxO3a did not interact in vitro or in vivo. HEK 293A cells were transfected with plasmids containing flag-FXR, HA-FoxO3a, HA or flag. Total cell lysates were isolated, and flag was pulled down by immunoprecipitation and subjected to immunoblot analysis for flag and HA. Input total lysates from transfected HEK 293A were used as positive controls for FoxO3a and FXR (A). Cytosol and nuclear fractions were obtained from transfected HEK 293A cells, and immunoblot analysis was performed for flag and FoxO3 (B). Age matched WT and FXR KO mice were treated with 4.5 g/kg ethanol or water by gavage for 16 h. FoxO3a in total liver lysates was pulled down by immunopreciptation and subjected to immunoblot analysis for FXR and FoxO3a (C).

    Article Snippet: Cell culture and transfection Human embryonic kidney 293A cells were cultured in DMEM medium with FBS and l -glutamate and transiently transfected with HA tagged FoxO3, Flag tagged FXR, HA or Flag plasmid constructs using TurboFect Transfection reagent (Thermo, Fisher Scientific) for 24 h. Total cell lysates then were extracted using HA cell lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA pH 7.5, 1 mM EGTA pH 7.5, 1% Triton X-100, protease inhibitors).

    Techniques: In Vitro, In Vivo, Transfection, Isolation, Immunoprecipitation, Mouse Assay

    Possible molecular and cellular events following acute ethanol treatment in FXR KO mouse livers. Acute ethanol exposure induces FoxO3a activation and mitophagy as a protective mechanism. FXR deficiency promotes Akt activation and subsequent serine 253 phosphorylation of FoxO3a, resulting in decreased nuclear FoxO3a retention and abolished transcription of autophagy related genes in response to ethanol. The defective hepatic autophagy in FXR KO mouse livers results in impaired mitophagy and compensatory induction of mitochondrial spheroid formation. Altogether, impaired FoxO3a-mediated autophagy and mitophagy in ethanol-treated FXR KO mouse livers lead to increased liver injury and steatosis.

    Journal: Redox Biology

    Article Title: Farnesoid X receptor regulates forkhead Box O3a activation in ethanol-induced autophagy and hepatotoxicity

    doi: 10.1016/j.redox.2014.08.007

    Figure Lengend Snippet: Possible molecular and cellular events following acute ethanol treatment in FXR KO mouse livers. Acute ethanol exposure induces FoxO3a activation and mitophagy as a protective mechanism. FXR deficiency promotes Akt activation and subsequent serine 253 phosphorylation of FoxO3a, resulting in decreased nuclear FoxO3a retention and abolished transcription of autophagy related genes in response to ethanol. The defective hepatic autophagy in FXR KO mouse livers results in impaired mitophagy and compensatory induction of mitochondrial spheroid formation. Altogether, impaired FoxO3a-mediated autophagy and mitophagy in ethanol-treated FXR KO mouse livers lead to increased liver injury and steatosis.

    Article Snippet: Cell culture and transfection Human embryonic kidney 293A cells were cultured in DMEM medium with FBS and l -glutamate and transiently transfected with HA tagged FoxO3, Flag tagged FXR, HA or Flag plasmid constructs using TurboFect Transfection reagent (Thermo, Fisher Scientific) for 24 h. Total cell lysates then were extracted using HA cell lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA pH 7.5, 1 mM EGTA pH 7.5, 1% Triton X-100, protease inhibitors).

    Techniques: Activation Assay