dual-luciferase assay Search Results


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  • 99
    Millipore dual luciferase assay mg63
    Ganoderma lucidum inhibits proliferation and induces apoptosis of <t>MG63</t> and U2-OS cells. (A, B) Viability of MG63 and U2-OS cells after treatment with various concentrations of G lucidum at different time points. (C, D) Cloning efficiencies of MG63 and U2-OS cells following treatment with 0, 100, or 200 µg/mL G lucidum for 12 days. (E, F) Analysis of apoptosis of MG63 and U2-OS cells after treatment with 0, 100, or 200 µg/mL G lucidum for 24 hours. (G, H) Changes in mRNA expression levels of Bax, Bad, caspase 3, and caspase 9 after treatment of MG63 and U2-OS cells with 0, 100, or 200 µg/mL G lucidum for 24 hours. N = 3, mean ± SD, * P
    Dual Luciferase Assay Mg63, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega dual luciferase assays dual luciferase assays
    Influence of LincRNA on the activity of the CYP46A1 gene promoter. Plasmids containing CYP46A1 rs754203 TT/CC and LincRNA RP11543C4.3 A/G were co-transfected into SH-SY5Y cells. <t>Dual</t> <t>luciferase</t> <t>assays</t> were performed. The ratio of Firefly luciferase to Renilla luciferase (A,B) and luciferase mRNA level (C,D) are shown. ∗∗∗ p
    Dual Luciferase Assays Dual Luciferase Assays, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3479 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega dual luciferase assay
    Wnt5a non-canonically regulates genes associated with cerebellar progenitor proliferation. ( A ) Schematic of TOPFlash and FOPFlash <t>luciferase</t> constructs. ( B ) Schematic represents the protocol followed for <t>dual</t> luciferase <t>assay</t> in primary cerebellar culture. ( C ) Dual luciferase assay with TOPFlash and FOPFlash luciferase reporter constructs. ( D ) Schematic shows the experimental procedure followed for gene expression analysis using real time PCR. ( E ) Real time PCR analysis of Cyclin D1, Sox2, Hes1 and Hes5 expression upon treatment with rWnt5a protein and non-canonical Wnt signaling blocker fumagillin. ( F ) Real time PCR analysis of cyclinD1 and Hes1 upon rWnt5a, DAPT and fumagillin treatment. ( G ) Schematic showing the pathway for activation of cyclin D1 by Wnt5a. ( H ) Schematic showing effect of Wnt5a loss during embryonic and postnatal stages on development of cerebellum. Data expressed as Mean ± SD, n = 3.
    Dual Luciferase Assay, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 10837 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genecopoeia dual luciferase assays
    Wnt5a non-canonically regulates genes associated with cerebellar progenitor proliferation. ( A ) Schematic of TOPFlash and FOPFlash <t>luciferase</t> constructs. ( B ) Schematic represents the protocol followed for <t>dual</t> luciferase <t>assay</t> in primary cerebellar culture. ( C ) Dual luciferase assay with TOPFlash and FOPFlash luciferase reporter constructs. ( D ) Schematic shows the experimental procedure followed for gene expression analysis using real time PCR. ( E ) Real time PCR analysis of Cyclin D1, Sox2, Hes1 and Hes5 expression upon treatment with rWnt5a protein and non-canonical Wnt signaling blocker fumagillin. ( F ) Real time PCR analysis of cyclinD1 and Hes1 upon rWnt5a, DAPT and fumagillin treatment. ( G ) Schematic showing the pathway for activation of cyclin D1 by Wnt5a. ( H ) Schematic showing effect of Wnt5a loss during embryonic and postnatal stages on development of cerebellum. Data expressed as Mean ± SD, n = 3.
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    Thermo Fisher dual luciferase assay
    Wnt5a non-canonically regulates genes associated with cerebellar progenitor proliferation. ( A ) Schematic of TOPFlash and FOPFlash <t>luciferase</t> constructs. ( B ) Schematic represents the protocol followed for <t>dual</t> luciferase <t>assay</t> in primary cerebellar culture. ( C ) Dual luciferase assay with TOPFlash and FOPFlash luciferase reporter constructs. ( D ) Schematic shows the experimental procedure followed for gene expression analysis using real time PCR. ( E ) Real time PCR analysis of Cyclin D1, Sox2, Hes1 and Hes5 expression upon treatment with rWnt5a protein and non-canonical Wnt signaling blocker fumagillin. ( F ) Real time PCR analysis of cyclinD1 and Hes1 upon rWnt5a, DAPT and fumagillin treatment. ( G ) Schematic showing the pathway for activation of cyclin D1 by Wnt5a. ( H ) Schematic showing effect of Wnt5a loss during embryonic and postnatal stages on development of cerebellum. Data expressed as Mean ± SD, n = 3.
    Dual Luciferase Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beyotime luciferase assay a dual luciferase assay kit
    Wnt5a non-canonically regulates genes associated with cerebellar progenitor proliferation. ( A ) Schematic of TOPFlash and FOPFlash <t>luciferase</t> constructs. ( B ) Schematic represents the protocol followed for <t>dual</t> luciferase <t>assay</t> in primary cerebellar culture. ( C ) Dual luciferase assay with TOPFlash and FOPFlash luciferase reporter constructs. ( D ) Schematic shows the experimental procedure followed for gene expression analysis using real time PCR. ( E ) Real time PCR analysis of Cyclin D1, Sox2, Hes1 and Hes5 expression upon treatment with rWnt5a protein and non-canonical Wnt signaling blocker fumagillin. ( F ) Real time PCR analysis of cyclinD1 and Hes1 upon rWnt5a, DAPT and fumagillin treatment. ( G ) Schematic showing the pathway for activation of cyclin D1 by Wnt5a. ( H ) Schematic showing effect of Wnt5a loss during embryonic and postnatal stages on development of cerebellum. Data expressed as Mean ± SD, n = 3.
    Luciferase Assay A Dual Luciferase Assay Kit, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genecopoeia dual luciferase assay kit
    Wnt5a non-canonically regulates genes associated with cerebellar progenitor proliferation. ( A ) Schematic of TOPFlash and FOPFlash <t>luciferase</t> constructs. ( B ) Schematic represents the protocol followed for <t>dual</t> luciferase <t>assay</t> in primary cerebellar culture. ( C ) Dual luciferase assay with TOPFlash and FOPFlash luciferase reporter constructs. ( D ) Schematic shows the experimental procedure followed for gene expression analysis using real time PCR. ( E ) Real time PCR analysis of Cyclin D1, Sox2, Hes1 and Hes5 expression upon treatment with rWnt5a protein and non-canonical Wnt signaling blocker fumagillin. ( F ) Real time PCR analysis of cyclinD1 and Hes1 upon rWnt5a, DAPT and fumagillin treatment. ( G ) Schematic showing the pathway for activation of cyclin D1 by Wnt5a. ( H ) Schematic showing effect of Wnt5a loss during embryonic and postnatal stages on development of cerebellum. Data expressed as Mean ± SD, n = 3.
    Dual Luciferase Assay Kit, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 92/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dual luciferase assay system
    Wnt5a non-canonically regulates genes associated with cerebellar progenitor proliferation. ( A ) Schematic of TOPFlash and FOPFlash <t>luciferase</t> constructs. ( B ) Schematic represents the protocol followed for <t>dual</t> luciferase <t>assay</t> in primary cerebellar culture. ( C ) Dual luciferase assay with TOPFlash and FOPFlash luciferase reporter constructs. ( D ) Schematic shows the experimental procedure followed for gene expression analysis using real time PCR. ( E ) Real time PCR analysis of Cyclin D1, Sox2, Hes1 and Hes5 expression upon treatment with rWnt5a protein and non-canonical Wnt signaling blocker fumagillin. ( F ) Real time PCR analysis of cyclinD1 and Hes1 upon rWnt5a, DAPT and fumagillin treatment. ( G ) Schematic showing the pathway for activation of cyclin D1 by Wnt5a. ( H ) Schematic showing effect of Wnt5a loss during embryonic and postnatal stages on development of cerebellum. Data expressed as Mean ± SD, n = 3.
    Dual Luciferase Assay System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TransGen biotech co dual luciferase assay system
    Wnt5a non-canonically regulates genes associated with cerebellar progenitor proliferation. ( A ) Schematic of TOPFlash and FOPFlash <t>luciferase</t> constructs. ( B ) Schematic represents the protocol followed for <t>dual</t> luciferase <t>assay</t> in primary cerebellar culture. ( C ) Dual luciferase assay with TOPFlash and FOPFlash luciferase reporter constructs. ( D ) Schematic shows the experimental procedure followed for gene expression analysis using real time PCR. ( E ) Real time PCR analysis of Cyclin D1, Sox2, Hes1 and Hes5 expression upon treatment with rWnt5a protein and non-canonical Wnt signaling blocker fumagillin. ( F ) Real time PCR analysis of cyclinD1 and Hes1 upon rWnt5a, DAPT and fumagillin treatment. ( G ) Schematic showing the pathway for activation of cyclin D1 by Wnt5a. ( H ) Schematic showing effect of Wnt5a loss during embryonic and postnatal stages on development of cerebellum. Data expressed as Mean ± SD, n = 3.
    Dual Luciferase Assay System, supplied by TransGen biotech co, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genecopoeia dual luciferase assay
    Wnt5a non-canonically regulates genes associated with cerebellar progenitor proliferation. ( A ) Schematic of TOPFlash and FOPFlash <t>luciferase</t> constructs. ( B ) Schematic represents the protocol followed for <t>dual</t> luciferase <t>assay</t> in primary cerebellar culture. ( C ) Dual luciferase assay with TOPFlash and FOPFlash luciferase reporter constructs. ( D ) Schematic shows the experimental procedure followed for gene expression analysis using real time PCR. ( E ) Real time PCR analysis of Cyclin D1, Sox2, Hes1 and Hes5 expression upon treatment with rWnt5a protein and non-canonical Wnt signaling blocker fumagillin. ( F ) Real time PCR analysis of cyclinD1 and Hes1 upon rWnt5a, DAPT and fumagillin treatment. ( G ) Schematic showing the pathway for activation of cyclin D1 by Wnt5a. ( H ) Schematic showing effect of Wnt5a loss during embryonic and postnatal stages on development of cerebellum. Data expressed as Mean ± SD, n = 3.
    Dual Luciferase Assay, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 92/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Ganoderma lucidum inhibits proliferation and induces apoptosis of MG63 and U2-OS cells. (A, B) Viability of MG63 and U2-OS cells after treatment with various concentrations of G lucidum at different time points. (C, D) Cloning efficiencies of MG63 and U2-OS cells following treatment with 0, 100, or 200 µg/mL G lucidum for 12 days. (E, F) Analysis of apoptosis of MG63 and U2-OS cells after treatment with 0, 100, or 200 µg/mL G lucidum for 24 hours. (G, H) Changes in mRNA expression levels of Bax, Bad, caspase 3, and caspase 9 after treatment of MG63 and U2-OS cells with 0, 100, or 200 µg/mL G lucidum for 24 hours. N = 3, mean ± SD, * P

    Journal: Integrative Cancer Therapies

    Article Title: Ganoderma lucidum Exerts an Anticancer Effect on Human Osteosarcoma Cells via Suppressing the Wnt/β-Catenin Signaling Pathway

    doi: 10.1177/1534735419890917

    Figure Lengend Snippet: Ganoderma lucidum inhibits proliferation and induces apoptosis of MG63 and U2-OS cells. (A, B) Viability of MG63 and U2-OS cells after treatment with various concentrations of G lucidum at different time points. (C, D) Cloning efficiencies of MG63 and U2-OS cells following treatment with 0, 100, or 200 µg/mL G lucidum for 12 days. (E, F) Analysis of apoptosis of MG63 and U2-OS cells after treatment with 0, 100, or 200 µg/mL G lucidum for 24 hours. (G, H) Changes in mRNA expression levels of Bax, Bad, caspase 3, and caspase 9 after treatment of MG63 and U2-OS cells with 0, 100, or 200 µg/mL G lucidum for 24 hours. N = 3, mean ± SD, * P

    Article Snippet: Dual-Luciferase Assay MG63 and U2-OS cells were, respectively, inoculated in a 96-well plate (5 × 103 cells/well) and incubated for 24 hours, then transfected with 200 ng TOP-Flash/FOP-Flash (Millipore, Burlington, MA), and 20 ng pRL-TK vector expressing Renilla luciferase (Promega, Madison, WI), following the recommended protocol using Lipofectamine 3000 (Thermo Scientific), for another 24 hours.

    Techniques: Clone Assay, Expressing

    Ganoderma lucidum inhibits the Wnt/β-catenin signaling pathway in MG63 and U2-OS cells. (A, B) Wnt/β-catenin signaling pathway reporter gene assay with or without pretreatment with CHIR-99021. Changes in TOP/FOP flash ratios of MG63 and U2-OS cells following treatment with 0, 100, or 200 µg/mL G lucidum for 24 hours, without pretreatment with CHIR-99021, N = 3, mean ± SD, * P

    Journal: Integrative Cancer Therapies

    Article Title: Ganoderma lucidum Exerts an Anticancer Effect on Human Osteosarcoma Cells via Suppressing the Wnt/β-Catenin Signaling Pathway

    doi: 10.1177/1534735419890917

    Figure Lengend Snippet: Ganoderma lucidum inhibits the Wnt/β-catenin signaling pathway in MG63 and U2-OS cells. (A, B) Wnt/β-catenin signaling pathway reporter gene assay with or without pretreatment with CHIR-99021. Changes in TOP/FOP flash ratios of MG63 and U2-OS cells following treatment with 0, 100, or 200 µg/mL G lucidum for 24 hours, without pretreatment with CHIR-99021, N = 3, mean ± SD, * P

    Article Snippet: Dual-Luciferase Assay MG63 and U2-OS cells were, respectively, inoculated in a 96-well plate (5 × 103 cells/well) and incubated for 24 hours, then transfected with 200 ng TOP-Flash/FOP-Flash (Millipore, Burlington, MA), and 20 ng pRL-TK vector expressing Renilla luciferase (Promega, Madison, WI), following the recommended protocol using Lipofectamine 3000 (Thermo Scientific), for another 24 hours.

    Techniques: Reporter Gene Assay

    Ganoderma lucidum suppresses the migration and invasion of MG63 and U2-OS cells. (A, B) Changes in migration of MG63 cells following treatment with 0, 100, or 200 µg/mL G lucidum for 24 or 48 hours. (C, D) Changes in migration of U2-OS cells following treatment with 0, 100, or 200 µg/mL G lucidum for 24 or 48 hours. (E, F) Changes in invasion of MG63 and U2-OS cells after treatment with 0, 100, or 200 µg/mL G lucidum for 24 hours. N = 3, mean ± SD, * P

    Journal: Integrative Cancer Therapies

    Article Title: Ganoderma lucidum Exerts an Anticancer Effect on Human Osteosarcoma Cells via Suppressing the Wnt/β-Catenin Signaling Pathway

    doi: 10.1177/1534735419890917

    Figure Lengend Snippet: Ganoderma lucidum suppresses the migration and invasion of MG63 and U2-OS cells. (A, B) Changes in migration of MG63 cells following treatment with 0, 100, or 200 µg/mL G lucidum for 24 or 48 hours. (C, D) Changes in migration of U2-OS cells following treatment with 0, 100, or 200 µg/mL G lucidum for 24 or 48 hours. (E, F) Changes in invasion of MG63 and U2-OS cells after treatment with 0, 100, or 200 µg/mL G lucidum for 24 hours. N = 3, mean ± SD, * P

    Article Snippet: Dual-Luciferase Assay MG63 and U2-OS cells were, respectively, inoculated in a 96-well plate (5 × 103 cells/well) and incubated for 24 hours, then transfected with 200 ng TOP-Flash/FOP-Flash (Millipore, Burlington, MA), and 20 ng pRL-TK vector expressing Renilla luciferase (Promega, Madison, WI), following the recommended protocol using Lipofectamine 3000 (Thermo Scientific), for another 24 hours.

    Techniques: Migration

    Cell proliferation, colony formation and xenograft tumor growth in CCT-NPC or parental cells. Notes: ( A ) MTT assays for acute exposure to Cd (1 nM, 1 µM and 1 mM). ( B ) MTT assays following 1 µM Cd treatment for 10 weeks. ( C ) Effects of chronic Cd exposure on the colonogenic ability in CNE-1/CNE-2 and CCT-CNE1/CCT-CNE2 cells (N=3). ( D ) Gross appearance of xenograft tumors at 28 days after CNE-1 or CCT-CNE1 cells injection. ( E ). Tumor growth curves and weight data in transplanted nude mice with CNE-1 and CCT-CNE1 through four weeks; data are mean (± SD) tumor volume (N=5). Each assay was performed in triplicate. * P

    Journal: Cancer Management and Research

    Article Title: Chronic cadmium exposure aggravates malignant phenotypes of nasopharyngeal carcinoma by activating the Wnt/β-catenin signaling pathway via hypermethylation of the casein kinase 1α promoter

    doi: 10.2147/CMAR.S171200

    Figure Lengend Snippet: Cell proliferation, colony formation and xenograft tumor growth in CCT-NPC or parental cells. Notes: ( A ) MTT assays for acute exposure to Cd (1 nM, 1 µM and 1 mM). ( B ) MTT assays following 1 µM Cd treatment for 10 weeks. ( C ) Effects of chronic Cd exposure on the colonogenic ability in CNE-1/CNE-2 and CCT-CNE1/CCT-CNE2 cells (N=3). ( D ) Gross appearance of xenograft tumors at 28 days after CNE-1 or CCT-CNE1 cells injection. ( E ). Tumor growth curves and weight data in transplanted nude mice with CNE-1 and CCT-CNE1 through four weeks; data are mean (± SD) tumor volume (N=5). Each assay was performed in triplicate. * P

    Article Snippet: Dual luciferase assay CNE-1 and CCT-CNE1 cells were seeded in 24-well plates overnight and then transiently transfected with TOPflash reporter plasmid (400 ng/well; Millipore, MA, USA) and Renilla luciferase plasmid (100 ng/well; Promega, Fitchburg, WI, USA) by Lipofectamine 3,000 (Invitrogen, Camarillo, CA, USA).

    Techniques: MTT Assay, Injection, Mouse Assay

    TLR2 and TLR4 expression is increased in response to SOD1 G93A overexpression. BV-2 cells were transiently transfected with CFP or SOD1 G93A -CFP plasmids and harvested at 8 or 24 h post-transfection. A , tlr2 mRNA expression in transfected BV-2 cells was measured at 8 h post-transfection by qPCR analysis, relative to control gene gapdh ( n = 8–10 wells pooled from 3 separate experiments; Kruskal–Wallis, Dunn’s multiple-comparison post hoc test). B , tlr4 mRNA was analyzed by qPCR analysis of BV-2 lysates 8 h post-transfection with CFP or SOD1 G93A -CFP ( n = 8–9 wells pooled from 2 separate experiments). Samples were normalized to internal control gapdh. C , E , TLR2 protein levels in CFP or SOD1 G93A -CFP-transfected BV-2 cells analyzed by Western blot. BV-2 cells were lysed in RIPA buffer 24 h post-transfection via nucleofection (normalized to α-Tubulin OD, n = 6 wells pooled from 4 separate experiments; p = 0.031, two-tailed paired t test). D , F , TLR4 protein levels in CFP or SOD1 G93A -CFP-transfected BV-2 cells analyzed by Western blot. BV-2 cells were lysed in RIPA buffer 24 h post-transfection via nucleofection (normalized to α-Tubulin OD n = 6 wells pooled from 4 separate experiments; p = 0.031, two-tailed paired t test). G , H , COX-II levels were assessed in SOD1 G93A overexpressing BV-2 cells ( n = 3 cultures from 2 separate platings; p = 0.016, two-tailed paired t test). BV-2 cells were transfected with CFP or SOD1 G93A -CFP via electroporation and lysed in RIPA buffer 24 h post-transfection and prepared for Western blot analysis. I , J , COX-II levels in TLR2- and TLR4 -inhibited BV-2 cells following stimulation with CFP or SOD1 G93A cMedia. BV-2 cells were treated with OxPAPC (30 µg/ml) simultaneous to cMedia treatment. Cells were lysed 24 h post-cMedia stimulation and prepared for Western blot analysis ( n = 3 wells, p = 0.002, one-way ANOVA, Tukey’s multiple comparison post hoc test). K , NF-κB activity in TLR4-deficient HEK293 and HEK293-TLR4-stably expressing cells stimulated with SOD1 G93A conditioned media. HEK293 and HEK293-TLR4 were cotransfected with κB-RE-luciferase and RLTK-Renilla-luciferase for normalization for 24 h and subsequently stimulated with CFP or SOD1 G93A cMedia for 8 or 24 h. The cells were lysed in passive lysis buffer and measured as κB-dependent firefly activity normalized to renilla luciferase activity per well ( n = 11 wells pooled from 2 separate experiments; p = 0.021, two-tailed paired t test between HEK-TLR4 treated cells). Conditioned media was generated by overexpression of CFP or SOD1 G93A -CFP vectors in NSC-34 cells, with serum-free conditioned media collected 3 d post-transfection.

    Journal: eNeuro

    Article Title: Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia 1-Induced Proinflammatory Signaling in Microglia 1 2-Induced Proinflammatory Signaling in Microglia 1 2 3

    doi: 10.1523/ENEURO.0099-15.2016

    Figure Lengend Snippet: TLR2 and TLR4 expression is increased in response to SOD1 G93A overexpression. BV-2 cells were transiently transfected with CFP or SOD1 G93A -CFP plasmids and harvested at 8 or 24 h post-transfection. A , tlr2 mRNA expression in transfected BV-2 cells was measured at 8 h post-transfection by qPCR analysis, relative to control gene gapdh ( n = 8–10 wells pooled from 3 separate experiments; Kruskal–Wallis, Dunn’s multiple-comparison post hoc test). B , tlr4 mRNA was analyzed by qPCR analysis of BV-2 lysates 8 h post-transfection with CFP or SOD1 G93A -CFP ( n = 8–9 wells pooled from 2 separate experiments). Samples were normalized to internal control gapdh. C , E , TLR2 protein levels in CFP or SOD1 G93A -CFP-transfected BV-2 cells analyzed by Western blot. BV-2 cells were lysed in RIPA buffer 24 h post-transfection via nucleofection (normalized to α-Tubulin OD, n = 6 wells pooled from 4 separate experiments; p = 0.031, two-tailed paired t test). D , F , TLR4 protein levels in CFP or SOD1 G93A -CFP-transfected BV-2 cells analyzed by Western blot. BV-2 cells were lysed in RIPA buffer 24 h post-transfection via nucleofection (normalized to α-Tubulin OD n = 6 wells pooled from 4 separate experiments; p = 0.031, two-tailed paired t test). G , H , COX-II levels were assessed in SOD1 G93A overexpressing BV-2 cells ( n = 3 cultures from 2 separate platings; p = 0.016, two-tailed paired t test). BV-2 cells were transfected with CFP or SOD1 G93A -CFP via electroporation and lysed in RIPA buffer 24 h post-transfection and prepared for Western blot analysis. I , J , COX-II levels in TLR2- and TLR4 -inhibited BV-2 cells following stimulation with CFP or SOD1 G93A cMedia. BV-2 cells were treated with OxPAPC (30 µg/ml) simultaneous to cMedia treatment. Cells were lysed 24 h post-cMedia stimulation and prepared for Western blot analysis ( n = 3 wells, p = 0.002, one-way ANOVA, Tukey’s multiple comparison post hoc test). K , NF-κB activity in TLR4-deficient HEK293 and HEK293-TLR4-stably expressing cells stimulated with SOD1 G93A conditioned media. HEK293 and HEK293-TLR4 were cotransfected with κB-RE-luciferase and RLTK-Renilla-luciferase for normalization for 24 h and subsequently stimulated with CFP or SOD1 G93A cMedia for 8 or 24 h. The cells were lysed in passive lysis buffer and measured as κB-dependent firefly activity normalized to renilla luciferase activity per well ( n = 11 wells pooled from 2 separate experiments; p = 0.021, two-tailed paired t test between HEK-TLR4 treated cells). Conditioned media was generated by overexpression of CFP or SOD1 G93A -CFP vectors in NSC-34 cells, with serum-free conditioned media collected 3 d post-transfection.

    Article Snippet: Dual-luciferase assay BV-2 cells were cotransfected by reverse transfection with renilla and firefly luciferase reporter gene vectors (renilla luciferase plasmid: firefly luciferase plasmid, 1:12) in Opti-MEM (Sigma-Aldrich; 1 µg plasmid per well/100 μl) using X-tremeGENE HP Reagent (Roche; 2 µl/µg plasmid).

    Techniques: Expressing, Over Expression, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Two Tailed Test, Electroporation, Activity Assay, Stable Transfection, Luciferase, Lysis, Generated

    Bid associates with TRAF6 in microglia and astrocytes, as shown by coimmunoprecipitation and PLA. A , Coimmunoprecipitation of Bid and TRAF6 in BV-2 cells. BV-2 cells were stimulated with LPS (1 µg/ml) for 15 min or 1 h and Bid was immunoprecipitated. Negative controls included anti-Bid immunoprecipitation from bid- deficient mixed glia lysates, and IgG immunoprecipitation from all samples. Cells were lysed in RIPA buffer and analyzed for TRAF6 content after immunoprecipitation of Bid. B , Coimmunoprecipitation of Bid and TRAF6 in BV-2 cells overexpressing TRAF6-FLAG. BV-2 cells were stimulated with LPS (1 µg/ml) and lysed in RIPA buffer. FLAG was detected by Western blotting and represents TRAF6FLAG immunoprecipitated with Bid in BV-2 cells. An IgG immunoprecipitation was included as a negative control. C , Coimmunoprecipitation of Bid and TRAF6 in WT and bid- deficient primary mixed glia stimulated with LPS for 1 and 4 h (100 ng/ml). Samples were lysed post-LPS stimulation and Bid was immunoprecipitated from the lysates. The samples were analyzed for TRAF6 content by Western blotting. IgG immunoprecipitation was carried out as an additional negative control. D , Coimmunoprecipitation of Bid and TRAF6 in wild-type and bid −/− astrocytes. Purified astrocytes were stimulated with LPS (100 ng/ml) for 1 and 4 h, and lysed in RIPA buffer for Bid immunoprecipitation. The samples were analyzed for TRAF6 by Western blotting. E , Representative images of PLA and phase contrast in TRAF6-FLAG overexpressing BV-2 cells immunostained with anti-Bid and anti-TRAF6 ( n = 2 wells/condition, 4 fields of view per well). Negative control representative images of PLA in TRAF6-FLAG overexpressing BV-2 cells immunostained with anti-TRAF6 and anti-HA-tag ( n = 1 well/condition, 6 fields of view-LPS, 1 field of view + LPS), or immunostained with anti-Bid and anti-IRF2 ( n = 1 well/condition, 7 fields of view). Scale bar, 10 µm. F , Quantification of PLA interactions in BV-2 cells. BV-2 cells were transfected with TRAF6-FLAG or empty FLAG vector and stimulated with LPS for 1 h. The cells were fixed with 3% paraformaldehyde, incubated with anti-Bid and anti-TRAF6 and PLA was quantified (significant increase of PLA dots in TRAF6 transfected versus control transfected cells and vehicle vs LPS treated cells, two-way ANOVA). Negative controls included immunostaining with anti-Bid plus anti-IRF2, and anti-TRAF6 plus anti-HA-tag.

    Journal: eNeuro

    Article Title: Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia 1-Induced Proinflammatory Signaling in Microglia 1 2-Induced Proinflammatory Signaling in Microglia 1 2 3

    doi: 10.1523/ENEURO.0099-15.2016

    Figure Lengend Snippet: Bid associates with TRAF6 in microglia and astrocytes, as shown by coimmunoprecipitation and PLA. A , Coimmunoprecipitation of Bid and TRAF6 in BV-2 cells. BV-2 cells were stimulated with LPS (1 µg/ml) for 15 min or 1 h and Bid was immunoprecipitated. Negative controls included anti-Bid immunoprecipitation from bid- deficient mixed glia lysates, and IgG immunoprecipitation from all samples. Cells were lysed in RIPA buffer and analyzed for TRAF6 content after immunoprecipitation of Bid. B , Coimmunoprecipitation of Bid and TRAF6 in BV-2 cells overexpressing TRAF6-FLAG. BV-2 cells were stimulated with LPS (1 µg/ml) and lysed in RIPA buffer. FLAG was detected by Western blotting and represents TRAF6FLAG immunoprecipitated with Bid in BV-2 cells. An IgG immunoprecipitation was included as a negative control. C , Coimmunoprecipitation of Bid and TRAF6 in WT and bid- deficient primary mixed glia stimulated with LPS for 1 and 4 h (100 ng/ml). Samples were lysed post-LPS stimulation and Bid was immunoprecipitated from the lysates. The samples were analyzed for TRAF6 content by Western blotting. IgG immunoprecipitation was carried out as an additional negative control. D , Coimmunoprecipitation of Bid and TRAF6 in wild-type and bid −/− astrocytes. Purified astrocytes were stimulated with LPS (100 ng/ml) for 1 and 4 h, and lysed in RIPA buffer for Bid immunoprecipitation. The samples were analyzed for TRAF6 by Western blotting. E , Representative images of PLA and phase contrast in TRAF6-FLAG overexpressing BV-2 cells immunostained with anti-Bid and anti-TRAF6 ( n = 2 wells/condition, 4 fields of view per well). Negative control representative images of PLA in TRAF6-FLAG overexpressing BV-2 cells immunostained with anti-TRAF6 and anti-HA-tag ( n = 1 well/condition, 6 fields of view-LPS, 1 field of view + LPS), or immunostained with anti-Bid and anti-IRF2 ( n = 1 well/condition, 7 fields of view). Scale bar, 10 µm. F , Quantification of PLA interactions in BV-2 cells. BV-2 cells were transfected with TRAF6-FLAG or empty FLAG vector and stimulated with LPS for 1 h. The cells were fixed with 3% paraformaldehyde, incubated with anti-Bid and anti-TRAF6 and PLA was quantified (significant increase of PLA dots in TRAF6 transfected versus control transfected cells and vehicle vs LPS treated cells, two-way ANOVA). Negative controls included immunostaining with anti-Bid plus anti-IRF2, and anti-TRAF6 plus anti-HA-tag.

    Article Snippet: Dual-luciferase assay BV-2 cells were cotransfected by reverse transfection with renilla and firefly luciferase reporter gene vectors (renilla luciferase plasmid: firefly luciferase plasmid, 1:12) in Opti-MEM (Sigma-Aldrich; 1 µg plasmid per well/100 μl) using X-tremeGENE HP Reagent (Roche; 2 µl/µg plasmid).

    Techniques: Proximity Ligation Assay, Immunoprecipitation, Western Blot, Negative Control, Purification, Transfection, Plasmid Preparation, Incubation, Immunostaining

    Reduced phosphorylation of IKKα/β, p65 and delayed IκBα degradation and reduced NF-κB activation in bid −/− microglia. A , Primary wild-type and bid −/− microglia were stimulated with LPS for 5–30 min in serum-free media before being fixed in 3% paraformaldehyde and stained with anti-phosphorylated IKKα/β (pIKKα/β) and anti-CD11b. Immunohistochemistry analysis of anti-pIKKα/β mean fluorescence on CD11b-positive cells is depicted. Scale bar, 50 µ m . B , C , Primary wild-type and bid −/− microglia were treated with LPS for 5, 15, 30 min, or 1 h in serum-free media before being lysed for Western blot analysis of pIKKα/β protein levels ( n = 3 pooled from 3 separate experiments; p = 0.014, 3-way ANOVA, Tukey post hoc test). D , E , Wild-type and bid −/− microglia were stimulated with LPS for 5 min to 2 h in serum-free media, lysed in RIPA buffer and IκBα levels were analyzed by Western blot ( n = 3–4 wells from 3–4 separate experiments). F , G , Wild-type and bid −/− microglia were stimulated with LPS for 1 h before being lysed in RIPA buffer. pp65 levels were assessed by Western blot ( n = 7 wells pooled from 6 separate experiments; p = 0.0162, one-way ANOVA, Tukey’s post hoc test). H , BV-2 cells were cotransfected with NF-κB-RE-luciferase and renilla-luciferase plasmids for 24 h and subsequently treated with LPS for 24 h. The cells were lysed in passive lysis buffer and NF-κB activation was quantified by dual luciferase assay (represented as relative κB-dependent firefly activity, n = 6–16 wells pooled from 2 separate experiments, 2 outliers removed, Grubbs test followed by Kruskal–Wallis and Dunn’s multiple-comparison post hoc test). I , J , COX-II levels in Bid-depleted BV-2 cells stimulated with CFP or SOD1 G93A cMedia. BV-2 cells were transfected with an siRNA targeting Bid (“siBid”) or a scrambled control siRNA (“siControl”), and stimulated with cMedia 48 h post-siRNA transfection, when Bid levels were optimally reduced. Twenty-four hours post-cMedia treatment the cells were lysed in RIPA and COX-II levels were measured. Dashed line indicated irrelevant lanes spliced out. Quantification of optical density was normalized to anti-α-tubulin for each Western blot ( n = 3, from 3 separate experiments).

    Journal: eNeuro

    Article Title: Bid Promotes K63-Linked Polyubiquitination of Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) and Sensitizes to Mutant SOD1-Induced Proinflammatory Signaling in Microglia 1-Induced Proinflammatory Signaling in Microglia 1 2-Induced Proinflammatory Signaling in Microglia 1 2 3

    doi: 10.1523/ENEURO.0099-15.2016

    Figure Lengend Snippet: Reduced phosphorylation of IKKα/β, p65 and delayed IκBα degradation and reduced NF-κB activation in bid −/− microglia. A , Primary wild-type and bid −/− microglia were stimulated with LPS for 5–30 min in serum-free media before being fixed in 3% paraformaldehyde and stained with anti-phosphorylated IKKα/β (pIKKα/β) and anti-CD11b. Immunohistochemistry analysis of anti-pIKKα/β mean fluorescence on CD11b-positive cells is depicted. Scale bar, 50 µ m . B , C , Primary wild-type and bid −/− microglia were treated with LPS for 5, 15, 30 min, or 1 h in serum-free media before being lysed for Western blot analysis of pIKKα/β protein levels ( n = 3 pooled from 3 separate experiments; p = 0.014, 3-way ANOVA, Tukey post hoc test). D , E , Wild-type and bid −/− microglia were stimulated with LPS for 5 min to 2 h in serum-free media, lysed in RIPA buffer and IκBα levels were analyzed by Western blot ( n = 3–4 wells from 3–4 separate experiments). F , G , Wild-type and bid −/− microglia were stimulated with LPS for 1 h before being lysed in RIPA buffer. pp65 levels were assessed by Western blot ( n = 7 wells pooled from 6 separate experiments; p = 0.0162, one-way ANOVA, Tukey’s post hoc test). H , BV-2 cells were cotransfected with NF-κB-RE-luciferase and renilla-luciferase plasmids for 24 h and subsequently treated with LPS for 24 h. The cells were lysed in passive lysis buffer and NF-κB activation was quantified by dual luciferase assay (represented as relative κB-dependent firefly activity, n = 6–16 wells pooled from 2 separate experiments, 2 outliers removed, Grubbs test followed by Kruskal–Wallis and Dunn’s multiple-comparison post hoc test). I , J , COX-II levels in Bid-depleted BV-2 cells stimulated with CFP or SOD1 G93A cMedia. BV-2 cells were transfected with an siRNA targeting Bid (“siBid”) or a scrambled control siRNA (“siControl”), and stimulated with cMedia 48 h post-siRNA transfection, when Bid levels were optimally reduced. Twenty-four hours post-cMedia treatment the cells were lysed in RIPA and COX-II levels were measured. Dashed line indicated irrelevant lanes spliced out. Quantification of optical density was normalized to anti-α-tubulin for each Western blot ( n = 3, from 3 separate experiments).

    Article Snippet: Dual-luciferase assay BV-2 cells were cotransfected by reverse transfection with renilla and firefly luciferase reporter gene vectors (renilla luciferase plasmid: firefly luciferase plasmid, 1:12) in Opti-MEM (Sigma-Aldrich; 1 µg plasmid per well/100 μl) using X-tremeGENE HP Reagent (Roche; 2 µl/µg plasmid).

    Techniques: Activation Assay, Staining, Immunohistochemistry, Fluorescence, Western Blot, Luciferase, Lysis, Activity Assay, Transfection

    Influence of LincRNA on the activity of the CYP46A1 gene promoter. Plasmids containing CYP46A1 rs754203 TT/CC and LincRNA RP11543C4.3 A/G were co-transfected into SH-SY5Y cells. Dual luciferase assays were performed. The ratio of Firefly luciferase to Renilla luciferase (A,B) and luciferase mRNA level (C,D) are shown. ∗∗∗ p

    Journal: Frontiers in Aging Neuroscience

    Article Title: LincRNA Plays a Role in the Effect of CYP46A1 Polymorphism in Alzheimer’s Disease – Related Pathology

    doi: 10.3389/fnagi.2019.00381

    Figure Lengend Snippet: Influence of LincRNA on the activity of the CYP46A1 gene promoter. Plasmids containing CYP46A1 rs754203 TT/CC and LincRNA RP11543C4.3 A/G were co-transfected into SH-SY5Y cells. Dual luciferase assays were performed. The ratio of Firefly luciferase to Renilla luciferase (A,B) and luciferase mRNA level (C,D) are shown. ∗∗∗ p

    Article Snippet: Dual Luciferase Assays Dual luciferase assays were performed by using the dual luciferase reporter assay system (Promega, United States) according to the manufacturer’s instructions.

    Techniques: Activity Assay, Transfection, Luciferase

    Exportin-5 knockdown leads to reduced silencing efficiency. ( A ) An outline of the experiment is illustrated. First the cells were transfected with different amounts of siExp5-1 and NegsiRNA and were incubated for 48 h. Then the cells were transfected with the dual luciferase reporter plasmids pGL2-Control (Firefly luciferase) and pRL-TK (Renilla luciferase) together with 1 nM siGL2 or NegsiRNA as a control for 24 h. ( B ) Dual luciferase results were obtained 24 h after transfection of the pGL2-Control (Firefly luciferase) and pRL-TK (Renilla luciferase) reporter plasmids together either with siGL2 or the NegsiRNA. Results were grouped together for each siRNA concentration used for 48 h. The ratios of target to control luciferase were normalized to the NegsiRNA control transfected for 48 h and for the luciferase assay indicated in black. The transfection of first the NegsiRNA for 48 h followed by siGL2 is shown in light grey, whereas the siExp5-1 transfection for 48 h followed by NegsiRNA is shown in dark grey and the siExp5-1 followed by siGL2 in white. ( C ) After 48 h total protein extracts from siExp5-1 transfected cells were immunoblotted with antibodies against Exp5 and GAPDH-HRP was used as a loading control. The plotted data were averaged from four independent experiments ±SD.

    Journal: Nucleic Acids Research

    Article Title: In situ fluorescence analysis demonstrates active siRNA exclusion from the nucleus by Exportin 5

    doi: 10.1093/nar/gkl001

    Figure Lengend Snippet: Exportin-5 knockdown leads to reduced silencing efficiency. ( A ) An outline of the experiment is illustrated. First the cells were transfected with different amounts of siExp5-1 and NegsiRNA and were incubated for 48 h. Then the cells were transfected with the dual luciferase reporter plasmids pGL2-Control (Firefly luciferase) and pRL-TK (Renilla luciferase) together with 1 nM siGL2 or NegsiRNA as a control for 24 h. ( B ) Dual luciferase results were obtained 24 h after transfection of the pGL2-Control (Firefly luciferase) and pRL-TK (Renilla luciferase) reporter plasmids together either with siGL2 or the NegsiRNA. Results were grouped together for each siRNA concentration used for 48 h. The ratios of target to control luciferase were normalized to the NegsiRNA control transfected for 48 h and for the luciferase assay indicated in black. The transfection of first the NegsiRNA for 48 h followed by siGL2 is shown in light grey, whereas the siExp5-1 transfection for 48 h followed by NegsiRNA is shown in dark grey and the siExp5-1 followed by siGL2 in white. ( C ) After 48 h total protein extracts from siExp5-1 transfected cells were immunoblotted with antibodies against Exp5 and GAPDH-HRP was used as a loading control. The plotted data were averaged from four independent experiments ±SD.

    Article Snippet: Luciferase assay Dual-luciferase assays (Promega GmbH) were performed 24–48 h after transfection according to the manufacturer's protocol for 24-well chambers and detected with a TD20/20 luminometer (Turner designs).

    Techniques: Transfection, Incubation, Luciferase, Concentration Assay

    A dual luciferase reporter assay validated the transcriptional activation of EjAP1 promoters by EjTFL1s, EjFD, and EjTFL1s-EjFD. (A) Schematic diagram of effector and reporter plasmid construction. (B) EjTFL1s, EjFD, and EjTFL1s-EjFD act on the EjAP1-1 promoter. (C) EjTFL1s, EjFD, and EjTFL1s-EjFD act on the EjAP1-2 promoter. The activation capabilities of EjTFL1s and EjFD were calculated by the ratio of LUC to REN. The ratio of empty vector LUC/REN was used as a control and set as 1. Error bars represent ± SE from three biological replicates. Asterisks indicate significantly different values compared with those for the empty vector (calculated by Student’s t -test), 0.0001

    Journal: Frontiers in Plant Science

    Article Title: EjTFL1 Genes Promote Growth but Inhibit Flower Bud Differentiation in Loquat

    doi: 10.3389/fpls.2020.00576

    Figure Lengend Snippet: A dual luciferase reporter assay validated the transcriptional activation of EjAP1 promoters by EjTFL1s, EjFD, and EjTFL1s-EjFD. (A) Schematic diagram of effector and reporter plasmid construction. (B) EjTFL1s, EjFD, and EjTFL1s-EjFD act on the EjAP1-1 promoter. (C) EjTFL1s, EjFD, and EjTFL1s-EjFD act on the EjAP1-2 promoter. The activation capabilities of EjTFL1s and EjFD were calculated by the ratio of LUC to REN. The ratio of empty vector LUC/REN was used as a control and set as 1. Error bars represent ± SE from three biological replicates. Asterisks indicate significantly different values compared with those for the empty vector (calculated by Student’s t -test), 0.0001

    Article Snippet: After 48–72 h, LUC and REN luciferases were quantified using the Dual Luciferase Assay kit (Promega, United States) with a Luminoskan Ascent Microplate Luminometer (Thermo, United States).

    Techniques: Luciferase, Reporter Assay, Activation Assay, Plasmid Preparation

    Wnt5a non-canonically regulates genes associated with cerebellar progenitor proliferation. ( A ) Schematic of TOPFlash and FOPFlash luciferase constructs. ( B ) Schematic represents the protocol followed for dual luciferase assay in primary cerebellar culture. ( C ) Dual luciferase assay with TOPFlash and FOPFlash luciferase reporter constructs. ( D ) Schematic shows the experimental procedure followed for gene expression analysis using real time PCR. ( E ) Real time PCR analysis of Cyclin D1, Sox2, Hes1 and Hes5 expression upon treatment with rWnt5a protein and non-canonical Wnt signaling blocker fumagillin. ( F ) Real time PCR analysis of cyclinD1 and Hes1 upon rWnt5a, DAPT and fumagillin treatment. ( G ) Schematic showing the pathway for activation of cyclin D1 by Wnt5a. ( H ) Schematic showing effect of Wnt5a loss during embryonic and postnatal stages on development of cerebellum. Data expressed as Mean ± SD, n = 3.

    Journal: Scientific Reports

    Article Title: Wnt5a is a crucial regulator of neurogenesis during cerebellum development

    doi: 10.1038/srep42523

    Figure Lengend Snippet: Wnt5a non-canonically regulates genes associated with cerebellar progenitor proliferation. ( A ) Schematic of TOPFlash and FOPFlash luciferase constructs. ( B ) Schematic represents the protocol followed for dual luciferase assay in primary cerebellar culture. ( C ) Dual luciferase assay with TOPFlash and FOPFlash luciferase reporter constructs. ( D ) Schematic shows the experimental procedure followed for gene expression analysis using real time PCR. ( E ) Real time PCR analysis of Cyclin D1, Sox2, Hes1 and Hes5 expression upon treatment with rWnt5a protein and non-canonical Wnt signaling blocker fumagillin. ( F ) Real time PCR analysis of cyclinD1 and Hes1 upon rWnt5a, DAPT and fumagillin treatment. ( G ) Schematic showing the pathway for activation of cyclin D1 by Wnt5a. ( H ) Schematic showing effect of Wnt5a loss during embryonic and postnatal stages on development of cerebellum. Data expressed as Mean ± SD, n = 3.

    Article Snippet: Dual-luciferase assay Dual Luciferase assay was done according to manufacturer’s protocol (Promega, Cat. no. E1910) using Luminometer (TD20/20, Promega).

    Techniques: Luciferase, Construct, Expressing, Real-time Polymerase Chain Reaction, Activation Assay