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  • 99
    Millipore luc activity
    Regulation of 4E-BP1 promoter activity by co-Smads. Firefly <t>LUC</t> activity normalized as in was assayed in different cell lines <t>co-transfected</t> with the −628/+219 4E-BP1 promoter fragment and various combinations of Smad2, 3, 4
    Luc Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 23 article reviews
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    99
    Promega luciferase reporter activities
    SOX2 modulates the expression of TUSC3 through miR-181a-5p and miR-30e-5p. a The sequence of the potential binding sites for miR-181a-5p and miR-30e-5p in the 3’-UTR of TUSC3. The sites are conserved in human and rodents. b The expression of TUSC3 is repressed by miR-181a-5p and miR-30e-5p mimics as measured by Western blot analysis. c The application of specific inhibitors against miR-181a-5p and miR-30e-5p increases the expression of TUSC3 in ZR7530 cells. d Schematic diagram of the binding sites for miR-181a-5p and miR-30e-5p in 3’-UTR of TUSC transcript. Mutated binding sites ( star ) were linked with <t>Luciferase</t> <t>reporter</t> (pMIR-Report-Luc). e Mutations of the potential binding sites for miR-181a-5p and miR-30e-5p lead to increased luciferase <t>activities</t> of TUSC3 reporter. Co-transfection of negative mimics and pMIR-Report-Luc-TUSC3-3UTR-WT construct is used as control (Con). Mic indicates miR-181a-5p or miR-30e-5p mimics. Mics indicates mixture of miR-181a-5p and miR-30e-5p mimics, ab* indicates both of a* and b* potential bindings sites are mutated, cd* indicates both of c* and d* potential bindings sites are mutated, and abcd* indicates all four potential bindings sites are mutated. f SOX2 knockdown leads to increased protein levels of TUSC3. g In xenograft initiated by ZR7530 cells, SOX2 knockdown leads to reduced expression of miR-181a-5p and miR-30e-5p, but not the expression of TUSC3 mRNA. The transcript levels were measured by examining RNA expression in three individual tumour nodules in each group. * indicates p
    Luciferase Reporter Activities, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 808 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luciferase reporter activities/product/Promega
    Average 99 stars, based on 808 article reviews
    Price from $9.99 to $1999.99
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    85
    Promega dual reporter luciferase activity
    SOX2 modulates the expression of TUSC3 through miR-181a-5p and miR-30e-5p. a The sequence of the potential binding sites for miR-181a-5p and miR-30e-5p in the 3’-UTR of TUSC3. The sites are conserved in human and rodents. b The expression of TUSC3 is repressed by miR-181a-5p and miR-30e-5p mimics as measured by Western blot analysis. c The application of specific inhibitors against miR-181a-5p and miR-30e-5p increases the expression of TUSC3 in ZR7530 cells. d Schematic diagram of the binding sites for miR-181a-5p and miR-30e-5p in 3’-UTR of TUSC transcript. Mutated binding sites ( star ) were linked with <t>Luciferase</t> <t>reporter</t> (pMIR-Report-Luc). e Mutations of the potential binding sites for miR-181a-5p and miR-30e-5p lead to increased luciferase <t>activities</t> of TUSC3 reporter. Co-transfection of negative mimics and pMIR-Report-Luc-TUSC3-3UTR-WT construct is used as control (Con). Mic indicates miR-181a-5p or miR-30e-5p mimics. Mics indicates mixture of miR-181a-5p and miR-30e-5p mimics, ab* indicates both of a* and b* potential bindings sites are mutated, cd* indicates both of c* and d* potential bindings sites are mutated, and abcd* indicates all four potential bindings sites are mutated. f SOX2 knockdown leads to increased protein levels of TUSC3. g In xenograft initiated by ZR7530 cells, SOX2 knockdown leads to reduced expression of miR-181a-5p and miR-30e-5p, but not the expression of TUSC3 mRNA. The transcript levels were measured by examining RNA expression in three individual tumour nodules in each group. * indicates p
    Dual Reporter Luciferase Activity, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dual reporter luciferase activity/product/Promega
    Average 85 stars, based on 4 article reviews
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    90
    Genecopoeia active renilla luciferase reporter
    SOX2 modulates the expression of TUSC3 through miR-181a-5p and miR-30e-5p. a The sequence of the potential binding sites for miR-181a-5p and miR-30e-5p in the 3’-UTR of TUSC3. The sites are conserved in human and rodents. b The expression of TUSC3 is repressed by miR-181a-5p and miR-30e-5p mimics as measured by Western blot analysis. c The application of specific inhibitors against miR-181a-5p and miR-30e-5p increases the expression of TUSC3 in ZR7530 cells. d Schematic diagram of the binding sites for miR-181a-5p and miR-30e-5p in 3’-UTR of TUSC transcript. Mutated binding sites ( star ) were linked with <t>Luciferase</t> <t>reporter</t> (pMIR-Report-Luc). e Mutations of the potential binding sites for miR-181a-5p and miR-30e-5p lead to increased luciferase <t>activities</t> of TUSC3 reporter. Co-transfection of negative mimics and pMIR-Report-Luc-TUSC3-3UTR-WT construct is used as control (Con). Mic indicates miR-181a-5p or miR-30e-5p mimics. Mics indicates mixture of miR-181a-5p and miR-30e-5p mimics, ab* indicates both of a* and b* potential bindings sites are mutated, cd* indicates both of c* and d* potential bindings sites are mutated, and abcd* indicates all four potential bindings sites are mutated. f SOX2 knockdown leads to increased protein levels of TUSC3. g In xenograft initiated by ZR7530 cells, SOX2 knockdown leads to reduced expression of miR-181a-5p and miR-30e-5p, but not the expression of TUSC3 mRNA. The transcript levels were measured by examining RNA expression in three individual tumour nodules in each group. * indicates p
    Active Renilla Luciferase Reporter, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 8 article reviews
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    94
    InvivoGen luciferase reporter
    SOX2 modulates the expression of TUSC3 through miR-181a-5p and miR-30e-5p. a The sequence of the potential binding sites for miR-181a-5p and miR-30e-5p in the 3’-UTR of TUSC3. The sites are conserved in human and rodents. b The expression of TUSC3 is repressed by miR-181a-5p and miR-30e-5p mimics as measured by Western blot analysis. c The application of specific inhibitors against miR-181a-5p and miR-30e-5p increases the expression of TUSC3 in ZR7530 cells. d Schematic diagram of the binding sites for miR-181a-5p and miR-30e-5p in 3’-UTR of TUSC transcript. Mutated binding sites ( star ) were linked with <t>Luciferase</t> <t>reporter</t> (pMIR-Report-Luc). e Mutations of the potential binding sites for miR-181a-5p and miR-30e-5p lead to increased luciferase <t>activities</t> of TUSC3 reporter. Co-transfection of negative mimics and pMIR-Report-Luc-TUSC3-3UTR-WT construct is used as control (Con). Mic indicates miR-181a-5p or miR-30e-5p mimics. Mics indicates mixture of miR-181a-5p and miR-30e-5p mimics, ab* indicates both of a* and b* potential bindings sites are mutated, cd* indicates both of c* and d* potential bindings sites are mutated, and abcd* indicates all four potential bindings sites are mutated. f SOX2 knockdown leads to increased protein levels of TUSC3. g In xenograft initiated by ZR7530 cells, SOX2 knockdown leads to reduced expression of miR-181a-5p and miR-30e-5p, but not the expression of TUSC3 mRNA. The transcript levels were measured by examining RNA expression in three individual tumour nodules in each group. * indicates p
    Luciferase Reporter, supplied by InvivoGen, used in various techniques. Bioz Stars score: 94/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luciferase reporter/product/InvivoGen
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    85
    Stratagene luciferase reporter pcre luc
    SOX2 modulates the expression of TUSC3 through miR-181a-5p and miR-30e-5p. a The sequence of the potential binding sites for miR-181a-5p and miR-30e-5p in the 3’-UTR of TUSC3. The sites are conserved in human and rodents. b The expression of TUSC3 is repressed by miR-181a-5p and miR-30e-5p mimics as measured by Western blot analysis. c The application of specific inhibitors against miR-181a-5p and miR-30e-5p increases the expression of TUSC3 in ZR7530 cells. d Schematic diagram of the binding sites for miR-181a-5p and miR-30e-5p in 3’-UTR of TUSC transcript. Mutated binding sites ( star ) were linked with <t>Luciferase</t> <t>reporter</t> (pMIR-Report-Luc). e Mutations of the potential binding sites for miR-181a-5p and miR-30e-5p lead to increased luciferase <t>activities</t> of TUSC3 reporter. Co-transfection of negative mimics and pMIR-Report-Luc-TUSC3-3UTR-WT construct is used as control (Con). Mic indicates miR-181a-5p or miR-30e-5p mimics. Mics indicates mixture of miR-181a-5p and miR-30e-5p mimics, ab* indicates both of a* and b* potential bindings sites are mutated, cd* indicates both of c* and d* potential bindings sites are mutated, and abcd* indicates all four potential bindings sites are mutated. f SOX2 knockdown leads to increased protein levels of TUSC3. g In xenograft initiated by ZR7530 cells, SOX2 knockdown leads to reduced expression of miR-181a-5p and miR-30e-5p, but not the expression of TUSC3 mRNA. The transcript levels were measured by examining RNA expression in three individual tumour nodules in each group. * indicates p
    Luciferase Reporter Pcre Luc, supplied by Stratagene, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luciferase reporter pcre luc/product/Stratagene
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    93
    InvivoGen luciferase reporter plasmid pnifty2 luc
    SOX2 modulates the expression of TUSC3 through miR-181a-5p and miR-30e-5p. a The sequence of the potential binding sites for miR-181a-5p and miR-30e-5p in the 3’-UTR of TUSC3. The sites are conserved in human and rodents. b The expression of TUSC3 is repressed by miR-181a-5p and miR-30e-5p mimics as measured by Western blot analysis. c The application of specific inhibitors against miR-181a-5p and miR-30e-5p increases the expression of TUSC3 in ZR7530 cells. d Schematic diagram of the binding sites for miR-181a-5p and miR-30e-5p in 3’-UTR of TUSC transcript. Mutated binding sites ( star ) were linked with <t>Luciferase</t> <t>reporter</t> (pMIR-Report-Luc). e Mutations of the potential binding sites for miR-181a-5p and miR-30e-5p lead to increased luciferase <t>activities</t> of TUSC3 reporter. Co-transfection of negative mimics and pMIR-Report-Luc-TUSC3-3UTR-WT construct is used as control (Con). Mic indicates miR-181a-5p or miR-30e-5p mimics. Mics indicates mixture of miR-181a-5p and miR-30e-5p mimics, ab* indicates both of a* and b* potential bindings sites are mutated, cd* indicates both of c* and d* potential bindings sites are mutated, and abcd* indicates all four potential bindings sites are mutated. f SOX2 knockdown leads to increased protein levels of TUSC3. g In xenograft initiated by ZR7530 cells, SOX2 knockdown leads to reduced expression of miR-181a-5p and miR-30e-5p, but not the expression of TUSC3 mRNA. The transcript levels were measured by examining RNA expression in three individual tumour nodules in each group. * indicates p
    Luciferase Reporter Plasmid Pnifty2 Luc, supplied by InvivoGen, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luciferase reporter plasmid pnifty2 luc/product/InvivoGen
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    Image Search Results


    Regulation of 4E-BP1 promoter activity by co-Smads. Firefly LUC activity normalized as in was assayed in different cell lines co-transfected with the −628/+219 4E-BP1 promoter fragment and various combinations of Smad2, 3, 4

    Journal: The EMBO Journal

    Article Title: 4E-BP1 is a target of Smad4 essential for TGF?-mediated inhibition of cell proliferation

    doi: 10.1038/emboj.2009.291

    Figure Lengend Snippet: Regulation of 4E-BP1 promoter activity by co-Smads. Firefly LUC activity normalized as in was assayed in different cell lines co-transfected with the −628/+219 4E-BP1 promoter fragment and various combinations of Smad2, 3, 4

    Article Snippet: Resistant clones were isolated, transiently transfected with pTRE-Tight-LUC (Clontech) and assayed for LUC activity following treatment with doxycycline (Sigma).

    Techniques: Activity Assay, Transfection

    IFN responsivness of the ISRE-promoter luciferase-reporter system. (A) Schematic representation of the vector construct (ISRE-CBG99) used to establish stable cell lines for monitoring ISRE activity driving a click beetle luciferase (CBL) reporter gene. (B) 2fTGH cells stably expressing the ISRE-CBG99 construct (2fTGH-ISRE-CBG99 cells) were treated with IFN-β (1000 U/ml for 0–14 h) and then monitored for luciferase-catalyzed luminescence over 0–4 h. Signal maximum was found at 50 min, and the optimal measurement window (OMW) with at least 90% preservation of signal) was found at 40–70 min. (C) Time course for effect of IFN-β (1000 U/ml) and IFN-γ (100 U/ml) on ISRE activity in 2fTGH-ISRE-CBG99 cells. (D) Corresponding time course for HEK293T-ISRE-CBG99 cells. (E) Ratio of ISRE activities when each cell line is treated with saturating concentrations of IFN-β versus IFN-γ for 0–24 h. * indicates significant differences between values for 2fTGH versus HEK293T cell lines. (F) Concentration-response curves for effect of IFN-β and IFN-γ on ISRE activity in 2fTGH-ISRE-CBG99 cells. (G) Corresponding concentration-response for HEK293T-ISRE-CBG99 cells.

    Journal: PLoS ONE

    Article Title: High Throughput Screening for Small Molecule Enhancers of the Interferon Signaling Pathway to Drive Next-Generation Antiviral Drug Discovery

    doi: 10.1371/journal.pone.0036594

    Figure Lengend Snippet: IFN responsivness of the ISRE-promoter luciferase-reporter system. (A) Schematic representation of the vector construct (ISRE-CBG99) used to establish stable cell lines for monitoring ISRE activity driving a click beetle luciferase (CBL) reporter gene. (B) 2fTGH cells stably expressing the ISRE-CBG99 construct (2fTGH-ISRE-CBG99 cells) were treated with IFN-β (1000 U/ml for 0–14 h) and then monitored for luciferase-catalyzed luminescence over 0–4 h. Signal maximum was found at 50 min, and the optimal measurement window (OMW) with at least 90% preservation of signal) was found at 40–70 min. (C) Time course for effect of IFN-β (1000 U/ml) and IFN-γ (100 U/ml) on ISRE activity in 2fTGH-ISRE-CBG99 cells. (D) Corresponding time course for HEK293T-ISRE-CBG99 cells. (E) Ratio of ISRE activities when each cell line is treated with saturating concentrations of IFN-β versus IFN-γ for 0–24 h. * indicates significant differences between values for 2fTGH versus HEK293T cell lines. (F) Concentration-response curves for effect of IFN-β and IFN-γ on ISRE activity in 2fTGH-ISRE-CBG99 cells. (G) Corresponding concentration-response for HEK293T-ISRE-CBG99 cells.

    Article Snippet: To determine whether compound effect depended on IFN production, the ISRE activity-luciferase reporter assay was also performed in the presence of mouse anti-human IFN-α/β receptor chain 2 (IFNAR2) blocking mAb (clone MMHAE-2; Millipore, Billerica, MA) at a concentration of 4 µg/ml.

    Techniques: Luciferase, Plasmid Preparation, Construct, Stable Transfection, Activity Assay, Expressing, Preserving, Concentration Assay

    Validation and specifcity of idarubicin capacity for ISRE activation. (A) Idarubicin concentration-response for ISRE activity without and with treatment with IFN-β (1, 5, and 15 U/ml). 2fTGH-ISRE-CBG99 cells were treated with idarubicin and IFN-β for 8 h. Overall significance for idarubicin dose P

    Journal: PLoS ONE

    Article Title: High Throughput Screening for Small Molecule Enhancers of the Interferon Signaling Pathway to Drive Next-Generation Antiviral Drug Discovery

    doi: 10.1371/journal.pone.0036594

    Figure Lengend Snippet: Validation and specifcity of idarubicin capacity for ISRE activation. (A) Idarubicin concentration-response for ISRE activity without and with treatment with IFN-β (1, 5, and 15 U/ml). 2fTGH-ISRE-CBG99 cells were treated with idarubicin and IFN-β for 8 h. Overall significance for idarubicin dose P

    Article Snippet: To determine whether compound effect depended on IFN production, the ISRE activity-luciferase reporter assay was also performed in the presence of mouse anti-human IFN-α/β receptor chain 2 (IFNAR2) blocking mAb (clone MMHAE-2; Millipore, Billerica, MA) at a concentration of 4 µg/ml.

    Techniques: Activation Assay, Concentration Assay, Activity Assay

    Effect of IFN-receptor blockade on idarubicin stimulation of ISRE activity. Idarubicin concentration-response for ISRE activity without and with treatment with IFN-β (1, 5, and15 U/ml) in the absence or presence of anti-IFNAR2 blocking mAb. *,† = p

    Journal: PLoS ONE

    Article Title: High Throughput Screening for Small Molecule Enhancers of the Interferon Signaling Pathway to Drive Next-Generation Antiviral Drug Discovery

    doi: 10.1371/journal.pone.0036594

    Figure Lengend Snippet: Effect of IFN-receptor blockade on idarubicin stimulation of ISRE activity. Idarubicin concentration-response for ISRE activity without and with treatment with IFN-β (1, 5, and15 U/ml) in the absence or presence of anti-IFNAR2 blocking mAb. *,† = p

    Article Snippet: To determine whether compound effect depended on IFN production, the ISRE activity-luciferase reporter assay was also performed in the presence of mouse anti-human IFN-α/β receptor chain 2 (IFNAR2) blocking mAb (clone MMHAE-2; Millipore, Billerica, MA) at a concentration of 4 µg/ml.

    Techniques: Activity Assay, Concentration Assay, Blocking Assay

    SOX2 modulates the expression of TUSC3 through miR-181a-5p and miR-30e-5p. a The sequence of the potential binding sites for miR-181a-5p and miR-30e-5p in the 3’-UTR of TUSC3. The sites are conserved in human and rodents. b The expression of TUSC3 is repressed by miR-181a-5p and miR-30e-5p mimics as measured by Western blot analysis. c The application of specific inhibitors against miR-181a-5p and miR-30e-5p increases the expression of TUSC3 in ZR7530 cells. d Schematic diagram of the binding sites for miR-181a-5p and miR-30e-5p in 3’-UTR of TUSC transcript. Mutated binding sites ( star ) were linked with Luciferase reporter (pMIR-Report-Luc). e Mutations of the potential binding sites for miR-181a-5p and miR-30e-5p lead to increased luciferase activities of TUSC3 reporter. Co-transfection of negative mimics and pMIR-Report-Luc-TUSC3-3UTR-WT construct is used as control (Con). Mic indicates miR-181a-5p or miR-30e-5p mimics. Mics indicates mixture of miR-181a-5p and miR-30e-5p mimics, ab* indicates both of a* and b* potential bindings sites are mutated, cd* indicates both of c* and d* potential bindings sites are mutated, and abcd* indicates all four potential bindings sites are mutated. f SOX2 knockdown leads to increased protein levels of TUSC3. g In xenograft initiated by ZR7530 cells, SOX2 knockdown leads to reduced expression of miR-181a-5p and miR-30e-5p, but not the expression of TUSC3 mRNA. The transcript levels were measured by examining RNA expression in three individual tumour nodules in each group. * indicates p

    Journal: Molecular Cancer

    Article Title: SOX2 regulates multiple malignant processes of breast cancer development through the SOX2/miR-181a-5p, miR-30e-5p/TUSC3 axis

    doi: 10.1186/s12943-017-0632-9

    Figure Lengend Snippet: SOX2 modulates the expression of TUSC3 through miR-181a-5p and miR-30e-5p. a The sequence of the potential binding sites for miR-181a-5p and miR-30e-5p in the 3’-UTR of TUSC3. The sites are conserved in human and rodents. b The expression of TUSC3 is repressed by miR-181a-5p and miR-30e-5p mimics as measured by Western blot analysis. c The application of specific inhibitors against miR-181a-5p and miR-30e-5p increases the expression of TUSC3 in ZR7530 cells. d Schematic diagram of the binding sites for miR-181a-5p and miR-30e-5p in 3’-UTR of TUSC transcript. Mutated binding sites ( star ) were linked with Luciferase reporter (pMIR-Report-Luc). e Mutations of the potential binding sites for miR-181a-5p and miR-30e-5p lead to increased luciferase activities of TUSC3 reporter. Co-transfection of negative mimics and pMIR-Report-Luc-TUSC3-3UTR-WT construct is used as control (Con). Mic indicates miR-181a-5p or miR-30e-5p mimics. Mics indicates mixture of miR-181a-5p and miR-30e-5p mimics, ab* indicates both of a* and b* potential bindings sites are mutated, cd* indicates both of c* and d* potential bindings sites are mutated, and abcd* indicates all four potential bindings sites are mutated. f SOX2 knockdown leads to increased protein levels of TUSC3. g In xenograft initiated by ZR7530 cells, SOX2 knockdown leads to reduced expression of miR-181a-5p and miR-30e-5p, but not the expression of TUSC3 mRNA. The transcript levels were measured by examining RNA expression in three individual tumour nodules in each group. * indicates p

    Article Snippet: Luciferase reporter assays Luciferase reporter activities were determined using the Dual-Luciferase Reporter Assay System (Promega, Cat No. E1910, Madison, USA).

    Techniques: Expressing, Sequencing, Binding Assay, Western Blot, Luciferase, Cotransfection, Construct, RNA Expression

    Mmp3 , Mmp10 , and Mmp13 are direct targets of murine miR-128, miR-134, and miR-330, respectively (A) Scheme for the potential binding site of miR-128, miR-134, and miR-330 in the 3′-UTR of Mmp3 , Mmp10 , and Mmp13 and the sequence of each intact miR-128, miR-134, and miR-330 binding site (wild-type, wt) and its mutant (Mut) within the luciferase reporter vector. (B) Luciferase assay with HEK293 cells, which were cotransfected with the indicated miRNA mimics (or control) and a luciferase reporter containing the 3′-UTR (wild type or mutant) of the indicated Mmps. An empty luciferase reporter construct was used as a negative control (Blank). Luciferase activities were measured 36 h post-transfection. miR-128, miR-134, and miR-330 suppressed the luciferase activity of Mmp3 , Mmp10 , and Mmp13 , respectively, in luciferase wild-type reporter constructs. The data are the mean ± SD for separate transfections (n = 3). * p

    Journal: Oncotarget

    Article Title: Up-regulation of matrix metalloproteinases in a mouse model of chemically induced colitis-associated cancer: the role of microRNAs

    doi:

    Figure Lengend Snippet: Mmp3 , Mmp10 , and Mmp13 are direct targets of murine miR-128, miR-134, and miR-330, respectively (A) Scheme for the potential binding site of miR-128, miR-134, and miR-330 in the 3′-UTR of Mmp3 , Mmp10 , and Mmp13 and the sequence of each intact miR-128, miR-134, and miR-330 binding site (wild-type, wt) and its mutant (Mut) within the luciferase reporter vector. (B) Luciferase assay with HEK293 cells, which were cotransfected with the indicated miRNA mimics (or control) and a luciferase reporter containing the 3′-UTR (wild type or mutant) of the indicated Mmps. An empty luciferase reporter construct was used as a negative control (Blank). Luciferase activities were measured 36 h post-transfection. miR-128, miR-134, and miR-330 suppressed the luciferase activity of Mmp3 , Mmp10 , and Mmp13 , respectively, in luciferase wild-type reporter constructs. The data are the mean ± SD for separate transfections (n = 3). * p

    Article Snippet: Luciferase reporter assay Luciferase activity was measured according to the dual-luciferase assay manual (Promega, Madison, WI, USA).

    Techniques: Binding Assay, Sequencing, Mutagenesis, Luciferase, Plasmid Preparation, Construct, Negative Control, Transfection, Activity Assay

    Ta CYP 81D5 confers salinity tolerance by promoting Zat12‐mediated ROS signalling pathway, thereby enhancing ROS scavenging. (a) The H 2 O 2 and (b) MDA contents in WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. (c) ROS levels of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines by carboxy‐H 2 DCFDA staining. Bar: 0.2 cm. (d) The abundance of Ta CAT , Ta APX , Ta NOX and Ta AOX transcript in roots of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. Ta EF 1‐ α (M90077) (Paolacci et al ., 2009 ) was chosen as the endogenous control. Relative transcript abundance of every gene in WT was calculated by giving the value 1. (e) CAT and (f) APX activities of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. (g) The transcript abundance of TaZat12 and Ta ZFP 36 in roots of SR 3 and JN 177. Relative transcript abundance of every gene in JN 177 was calculated by giving the value 1. (h) The transcript abundance of TaZat12 and Ta ZFP 36 in roots of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. Relative transcript abundance of every gene in WT was calculated by giving the value 1. (i) Y1H assay showing that TaZat12 could bind the A(G/C)T repeats element in Ta APX promoter. Ta APX ‐P : the ~500‐bp promoter fragment of Ta APX ; Ta APX ‐ mP : a fragment deleting the A(G/C)T repeats element; SD /‐Leu, SD medium without Leu; SD /‐Leu/AbA 400 , SD medium without Leu supplemented with AbA at the concentration of 400 ng/mL. Transformed yeast cells were dotted at 10 −1 dilutions on the selective medium. (j) Transient expression assay showing that TaZat12 could activate the expression of Ta APX , and the A(G/C)T repeats element is essential for this activation. The black column represents the relative LUC / REN ratio of the reporter in the present of empty effector ( pBI 221 empty vector) without TaZat12; and the grey column represents the relative LUC / REN ratio of the reporter in the present of effector containing TaZat12. Data are presented as mean ± SE of at least three biological replicates. Columns marked with one asterisk indicate significant differences ( P

    Journal: Plant Biotechnology Journal

    Article Title: TaCYP81D5, one member in a wheat cytochrome P450 gene cluster, confers salinity tolerance via reactive oxygen species scavenging

    doi: 10.1111/pbi.13247

    Figure Lengend Snippet: Ta CYP 81D5 confers salinity tolerance by promoting Zat12‐mediated ROS signalling pathway, thereby enhancing ROS scavenging. (a) The H 2 O 2 and (b) MDA contents in WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. (c) ROS levels of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines by carboxy‐H 2 DCFDA staining. Bar: 0.2 cm. (d) The abundance of Ta CAT , Ta APX , Ta NOX and Ta AOX transcript in roots of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. Ta EF 1‐ α (M90077) (Paolacci et al ., 2009 ) was chosen as the endogenous control. Relative transcript abundance of every gene in WT was calculated by giving the value 1. (e) CAT and (f) APX activities of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. (g) The transcript abundance of TaZat12 and Ta ZFP 36 in roots of SR 3 and JN 177. Relative transcript abundance of every gene in JN 177 was calculated by giving the value 1. (h) The transcript abundance of TaZat12 and Ta ZFP 36 in roots of WT , Ta OE ‐null, Ta OE 1 and Ta OE 2 wheat lines. Relative transcript abundance of every gene in WT was calculated by giving the value 1. (i) Y1H assay showing that TaZat12 could bind the A(G/C)T repeats element in Ta APX promoter. Ta APX ‐P : the ~500‐bp promoter fragment of Ta APX ; Ta APX ‐ mP : a fragment deleting the A(G/C)T repeats element; SD /‐Leu, SD medium without Leu; SD /‐Leu/AbA 400 , SD medium without Leu supplemented with AbA at the concentration of 400 ng/mL. Transformed yeast cells were dotted at 10 −1 dilutions on the selective medium. (j) Transient expression assay showing that TaZat12 could activate the expression of Ta APX , and the A(G/C)T repeats element is essential for this activation. The black column represents the relative LUC / REN ratio of the reporter in the present of empty effector ( pBI 221 empty vector) without TaZat12; and the grey column represents the relative LUC / REN ratio of the reporter in the present of effector containing TaZat12. Data are presented as mean ± SE of at least three biological replicates. Columns marked with one asterisk indicate significant differences ( P

    Article Snippet: The relative LUC activity was determined by LUC/REN ratio using Dual‐Luciferase Reporter Assay Kit (Promega, Madison, WI) and a Synergy 2 multimode microplate (BioTek, Winooski, VT) according to the manufacturer's instructions.

    Techniques: Multiple Displacement Amplification, Staining, Y1H Assay, Concentration Assay, Transformation Assay, Expressing, Activation Assay, Plasmid Preparation

    The RNF31 RBR domain is required for association with and stabilization of ERα. ( a ) RNF31 domain structure and deletion mutants used in this study (full-length, ΔUBA and ΔRBR). ZnF_RBZ, putative zinc-finger ubiquitin-binding domain; UBA, ubiquitin-associated domain, mediates interaction with RBCK1/LUBAC; RING-IBR-RING (RBR), atypical E3 ubiquitin ligase domain. ( b ) RNF31 association with ERα requires the RBR domain. HEK-293 cells were transfected with ERα together with plasmids expressing Myc-tagged full-length RNF31, variants deleting the UBA or RBR domains, respectively, or the Myc-tag alone. Cells were harvested 24 h after transfection and whole-cell extracts were prepared for co-IP. The predicted molecular weights of the RNF31 derivatives and ERα are indicated. ( c ) The RBR domain is necessary for the RNF31-mediated increase of endogenous ERα protein levels. MCF-7 cells were transfected with plasmids expressing Myc-tagged RNF31 derivatives or the Myc-tag alone, as indicated. 48 h after transfection, whole-cell extracts were prepared and levels of ERα protein assayed by western blot analysis. The predicted molecular weights of RNF31 variants, ERα and the loading control GAPDH are indicated. ( d ) The RBR domain of RNF31 is required to increase ERα signaling. MCF-7 cells were transfected with plasmids expressing Myc-tagged RNF31 derivatives, or the Myc-tag alone, as indicated, along with an ERE-luciferase reporter plasmid. Luciferase activity was measured 48 h after transfection and calculated from experiments performed in triplicates. Data are shown as mean±s.d. ( n =3). *** P

    Journal: Oncogene

    Article Title: The atypical ubiquitin ligase RNF31 stabilizes estrogen receptor α and modulates estrogen-stimulated breast cancer cell proliferation

    doi: 10.1038/onc.2013.573

    Figure Lengend Snippet: The RNF31 RBR domain is required for association with and stabilization of ERα. ( a ) RNF31 domain structure and deletion mutants used in this study (full-length, ΔUBA and ΔRBR). ZnF_RBZ, putative zinc-finger ubiquitin-binding domain; UBA, ubiquitin-associated domain, mediates interaction with RBCK1/LUBAC; RING-IBR-RING (RBR), atypical E3 ubiquitin ligase domain. ( b ) RNF31 association with ERα requires the RBR domain. HEK-293 cells were transfected with ERα together with plasmids expressing Myc-tagged full-length RNF31, variants deleting the UBA or RBR domains, respectively, or the Myc-tag alone. Cells were harvested 24 h after transfection and whole-cell extracts were prepared for co-IP. The predicted molecular weights of the RNF31 derivatives and ERα are indicated. ( c ) The RBR domain is necessary for the RNF31-mediated increase of endogenous ERα protein levels. MCF-7 cells were transfected with plasmids expressing Myc-tagged RNF31 derivatives or the Myc-tag alone, as indicated. 48 h after transfection, whole-cell extracts were prepared and levels of ERα protein assayed by western blot analysis. The predicted molecular weights of RNF31 variants, ERα and the loading control GAPDH are indicated. ( d ) The RBR domain of RNF31 is required to increase ERα signaling. MCF-7 cells were transfected with plasmids expressing Myc-tagged RNF31 derivatives, or the Myc-tag alone, as indicated, along with an ERE-luciferase reporter plasmid. Luciferase activity was measured 48 h after transfection and calculated from experiments performed in triplicates. Data are shown as mean±s.d. ( n =3). *** P

    Article Snippet: Luciferase reporter assays Luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega, Mannheim, Germany).

    Techniques: Binding Assay, Transfection, Expressing, Co-Immunoprecipitation Assay, Western Blot, Luciferase, Plasmid Preparation, Activity Assay

    RNF31 depletion decreases ERα protein levels and ERα signaling. ( a ) RNF31 depletion reduces ERα protein levels. MCF-7 cells were transfected with siRNF31 or siControl and treated with 10 n M E2 or vehicle for 72 h. ERα and RNF31 levels were determined by western blot analysis. GAPDH was used as internal control. ( b ) RNF31 depletion or overexpression affects ERα-dependent expression of an ERE-luciferase reporter gene. MCF-7 cells were transfected with siRNF31 or siControl or with plasmids expressing Myc-tagged RNF31 or Myc-tag vector alone or together with the ERE reporter plasmid. Subsequently, cells were treated with 10 n M E2 or vehicle. Luciferase activity was measured 48 h after transfection. Shown are data from triplicate measurements. *** P

    Journal: Oncogene

    Article Title: The atypical ubiquitin ligase RNF31 stabilizes estrogen receptor α and modulates estrogen-stimulated breast cancer cell proliferation

    doi: 10.1038/onc.2013.573

    Figure Lengend Snippet: RNF31 depletion decreases ERα protein levels and ERα signaling. ( a ) RNF31 depletion reduces ERα protein levels. MCF-7 cells were transfected with siRNF31 or siControl and treated with 10 n M E2 or vehicle for 72 h. ERα and RNF31 levels were determined by western blot analysis. GAPDH was used as internal control. ( b ) RNF31 depletion or overexpression affects ERα-dependent expression of an ERE-luciferase reporter gene. MCF-7 cells were transfected with siRNF31 or siControl or with plasmids expressing Myc-tagged RNF31 or Myc-tag vector alone or together with the ERE reporter plasmid. Subsequently, cells were treated with 10 n M E2 or vehicle. Luciferase activity was measured 48 h after transfection. Shown are data from triplicate measurements. *** P

    Article Snippet: Luciferase reporter assays Luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega, Mannheim, Germany).

    Techniques: Transfection, Western Blot, Over Expression, Expressing, Luciferase, Plasmid Preparation, Activity Assay

    The ubiquitin ligase activity of RNF31 is required for ERα stabilization, signaling and mono-ubiquitination. ( a ) The mono-ubiquitination function of RNF31 is required for the RNF31-mediated increase of endogenous ERα protein levels. MCF-7 cells were transfected with plasmids expressing Myc-tagged RNF31, Myc-tagged RNF31 R1/2M, in which cysteine residues responsible for the transfer of ubiquitin to substrates have been mutated or the Myc-tag alone, as indicated. Forty-eight hours after transfection, whole-cell extracts were prepared and levels of ERα protein assayed by western blot analysis. The predicted molecular weights of RNF31 variants, ERα and the loading control GAPDH are indicated. ( b ) The mono-ubiquitination function of RNF31 is required to increase ERα signaling. MCF-7 cells were transfected with plasmids expressing Myc-tagged RNF31, Myc-tagged RNF31 R1/2M or the Myc-tag alone, as indicated, along with an ERE-luciferase reporter plasmid. Luciferase activity was measured 48 h after transfection and calculated from experiments performed in triplicates. Data are shown as mean±s.d. ( n =3). *** P

    Journal: Oncogene

    Article Title: The atypical ubiquitin ligase RNF31 stabilizes estrogen receptor α and modulates estrogen-stimulated breast cancer cell proliferation

    doi: 10.1038/onc.2013.573

    Figure Lengend Snippet: The ubiquitin ligase activity of RNF31 is required for ERα stabilization, signaling and mono-ubiquitination. ( a ) The mono-ubiquitination function of RNF31 is required for the RNF31-mediated increase of endogenous ERα protein levels. MCF-7 cells were transfected with plasmids expressing Myc-tagged RNF31, Myc-tagged RNF31 R1/2M, in which cysteine residues responsible for the transfer of ubiquitin to substrates have been mutated or the Myc-tag alone, as indicated. Forty-eight hours after transfection, whole-cell extracts were prepared and levels of ERα protein assayed by western blot analysis. The predicted molecular weights of RNF31 variants, ERα and the loading control GAPDH are indicated. ( b ) The mono-ubiquitination function of RNF31 is required to increase ERα signaling. MCF-7 cells were transfected with plasmids expressing Myc-tagged RNF31, Myc-tagged RNF31 R1/2M or the Myc-tag alone, as indicated, along with an ERE-luciferase reporter plasmid. Luciferase activity was measured 48 h after transfection and calculated from experiments performed in triplicates. Data are shown as mean±s.d. ( n =3). *** P

    Article Snippet: Luciferase reporter assays Luciferase activity was measured using the Dual-Luciferase Reporter Assay (Promega, Mannheim, Germany).

    Techniques: Activity Assay, Transfection, Expressing, Western Blot, Luciferase, Plasmid Preparation

    The 3'-UTR of CDK5R1 causes a decrease in luciferase activity . A) Schematic representation of luciferase construct carrying the human 3'-UTR of CDK5R1 . The pGL4.71P control construct does not contain CDK5R1 UTR sequences. The Renilla luciferase gene is indicated by the grey bars, and the region of the 3'-UTR is represented by the white bars. The numbers on the right indicate the 3'-UTR nucleotides included in the chimeric construct. Numbering starts from the first nucleotide beyond the stop codon for CDK5R1 . Transcription was under the control of the SV40 promoter (hatched bars) and the polyA site (black bars). B) Luciferase activity of pGL4.71P-UTR construct in SK-N-BE, SH-SY5Y, HEK-293 and MCF-7 cell lines assessed using the Dual-Glo Luciferase assay system. Cells were transiently co-transfected with the pGL4.71P-UTR construct (Renilla luciferase) and the pGL3 (Firefly luciferase) vector and were harvested 24 hours post-transfection (see Methods). Luciferase activity of the chimeric reporter gene, normalized for transfection efficiency against the Firefly luciferase activity, is represented as a percentage of the activity observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase values were obtained from at least four independent experiments (* p

    Journal: BMC Molecular Biology

    Article Title: The 3' untranslated region of human Cyclin-Dependent Kinase 5 Regulatory subunit 1 contains regulatory elements affecting transcript stability

    doi: 10.1186/1471-2199-8-111

    Figure Lengend Snippet: The 3'-UTR of CDK5R1 causes a decrease in luciferase activity . A) Schematic representation of luciferase construct carrying the human 3'-UTR of CDK5R1 . The pGL4.71P control construct does not contain CDK5R1 UTR sequences. The Renilla luciferase gene is indicated by the grey bars, and the region of the 3'-UTR is represented by the white bars. The numbers on the right indicate the 3'-UTR nucleotides included in the chimeric construct. Numbering starts from the first nucleotide beyond the stop codon for CDK5R1 . Transcription was under the control of the SV40 promoter (hatched bars) and the polyA site (black bars). B) Luciferase activity of pGL4.71P-UTR construct in SK-N-BE, SH-SY5Y, HEK-293 and MCF-7 cell lines assessed using the Dual-Glo Luciferase assay system. Cells were transiently co-transfected with the pGL4.71P-UTR construct (Renilla luciferase) and the pGL3 (Firefly luciferase) vector and were harvested 24 hours post-transfection (see Methods). Luciferase activity of the chimeric reporter gene, normalized for transfection efficiency against the Firefly luciferase activity, is represented as a percentage of the activity observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase values were obtained from at least four independent experiments (* p

    Article Snippet: Measurement of luciferase activity Luciferase reporter assays were performed using the Dual-Glo Luciferase Reporter Assay System (Promega).

    Techniques: Luciferase, Activity Assay, Construct, Transfection, Plasmid Preparation

    CDK5R1 3'-UTR fragments differently affect reporter gene expression . A) Schematic representation of luciferase constructs carrying fragments of the 3'-UTR of CDK5R1 . The pGL4.71P control construct contained no CDK5R1 UTR sequences. The chimeric constructs were created by cloning C1, C2, C3, C4, C5 and C6 3'-UTR fragments downstream of the Renilla luciferase gene. B) Luciferase activity of the six pGL4.71P- constructs in SK-N-BE, SH-SY5Y, HEK-293 and MCF-7 cell lines. Cells, transiently co-transfected with the pGL4.71P- constructs (Renilla luciferase) and the pGL3 (Firefly luciferase) vector were harvested 24 hours post-transfection. Luciferase activity of the chimeric constructs normalized as described, is represented as a percentage of the activity observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase values were obtained from at least four independent experiments (* p

    Journal: BMC Molecular Biology

    Article Title: The 3' untranslated region of human Cyclin-Dependent Kinase 5 Regulatory subunit 1 contains regulatory elements affecting transcript stability

    doi: 10.1186/1471-2199-8-111

    Figure Lengend Snippet: CDK5R1 3'-UTR fragments differently affect reporter gene expression . A) Schematic representation of luciferase constructs carrying fragments of the 3'-UTR of CDK5R1 . The pGL4.71P control construct contained no CDK5R1 UTR sequences. The chimeric constructs were created by cloning C1, C2, C3, C4, C5 and C6 3'-UTR fragments downstream of the Renilla luciferase gene. B) Luciferase activity of the six pGL4.71P- constructs in SK-N-BE, SH-SY5Y, HEK-293 and MCF-7 cell lines. Cells, transiently co-transfected with the pGL4.71P- constructs (Renilla luciferase) and the pGL3 (Firefly luciferase) vector were harvested 24 hours post-transfection. Luciferase activity of the chimeric constructs normalized as described, is represented as a percentage of the activity observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase values were obtained from at least four independent experiments (* p

    Article Snippet: Measurement of luciferase activity Luciferase reporter assays were performed using the Dual-Glo Luciferase Reporter Assay System (Promega).

    Techniques: Expressing, Luciferase, Construct, Clone Assay, Activity Assay, Transfection, Plasmid Preparation

    Decrease in steady state mRNA levels is due to altered rates of messenger degradation which is not ARE dependent . A) Decay of pGL4.71P-C2 mRNA (black squares) compared with that of pGL4.71P control (black diamonds). SK-N-BE cells were transiently transfected with reporter gene constructs and the pGL3 (firefly luciferase) vector. 24 hours after transfection (time = 0 h), cells were treated with DRB. Total RNA was extracted after various time-points. RNA was reverse-transcribed and RealTime RT-PCR was performed. Renilla luciferase transcript levels were normalized to GAPDH and firefly luciferase mRNAs. Data are expressed as percentage of RNA remaining after DRB addition and are representative of three independent experiments. B) Schematic representation of luciferase constructs carrying C2 fragment and sub-fragments. C) Luciferase activity of the chimeric constructs in SK-N-BE, SH-SY5Y, HEK-293 and MCF-7 cell lines. Cells, transiently co-transfected with the pGL4.71P- constructs (Renilla luciferase) and the pGL3 (Firefly luciferase) vector were harvested 24 hours post-transfection. Luciferase activity of the chimeric constructs normalized as described, is represented as a percentage of the activity observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase values were obtained from at least four independent experiments (* p

    Journal: BMC Molecular Biology

    Article Title: The 3' untranslated region of human Cyclin-Dependent Kinase 5 Regulatory subunit 1 contains regulatory elements affecting transcript stability

    doi: 10.1186/1471-2199-8-111

    Figure Lengend Snippet: Decrease in steady state mRNA levels is due to altered rates of messenger degradation which is not ARE dependent . A) Decay of pGL4.71P-C2 mRNA (black squares) compared with that of pGL4.71P control (black diamonds). SK-N-BE cells were transiently transfected with reporter gene constructs and the pGL3 (firefly luciferase) vector. 24 hours after transfection (time = 0 h), cells were treated with DRB. Total RNA was extracted after various time-points. RNA was reverse-transcribed and RealTime RT-PCR was performed. Renilla luciferase transcript levels were normalized to GAPDH and firefly luciferase mRNAs. Data are expressed as percentage of RNA remaining after DRB addition and are representative of three independent experiments. B) Schematic representation of luciferase constructs carrying C2 fragment and sub-fragments. C) Luciferase activity of the chimeric constructs in SK-N-BE, SH-SY5Y, HEK-293 and MCF-7 cell lines. Cells, transiently co-transfected with the pGL4.71P- constructs (Renilla luciferase) and the pGL3 (Firefly luciferase) vector were harvested 24 hours post-transfection. Luciferase activity of the chimeric constructs normalized as described, is represented as a percentage of the activity observed in cells transfected with pGL4.71P (defined as 100%). Means ± s.d. luciferase values were obtained from at least four independent experiments (* p

    Article Snippet: Measurement of luciferase activity Luciferase reporter assays were performed using the Dual-Glo Luciferase Reporter Assay System (Promega).

    Techniques: Transfection, Construct, Luciferase, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Activity Assay

    LiCl regulates the expression of Six2 at mRNA and protein level. ( A ) pGL3- Six2 promoter-Luciferase construction was simulate by diagram. The Six2 promoter ranging from −2322 to −323 (Six genome sequence) was obtained from NCBI; ( B ) HEK293T cells were co-tra nsfected with pRL-SV40 (renilla control) and pGL3- Six2 promoter-LuC for 36 h. Luciferase activity was normalized to Renilla control. p -values were calculated by Student t -test. Values represents mean values ± SEM of triplicate experiments, *** p

    Journal: International Journal of Molecular Sciences

    Article Title: Six2 Is a Coordinator of LiCl-Induced Cell Proliferation and Apoptosis

    doi: 10.3390/ijms17091504

    Figure Lengend Snippet: LiCl regulates the expression of Six2 at mRNA and protein level. ( A ) pGL3- Six2 promoter-Luciferase construction was simulate by diagram. The Six2 promoter ranging from −2322 to −323 (Six genome sequence) was obtained from NCBI; ( B ) HEK293T cells were co-tra nsfected with pRL-SV40 (renilla control) and pGL3- Six2 promoter-LuC for 36 h. Luciferase activity was normalized to Renilla control. p -values were calculated by Student t -test. Values represents mean values ± SEM of triplicate experiments, *** p

    Article Snippet: For luciferase assays, HEK293T cells were transfected with pGL3-Six2 promoter-luciferase (500 ng/well) and pRL-SV40 (10 ng/well) for 36 h and then treated with LiCl of increasing dosages for 12 h. Luciferase (Luc) activity was assayed using Dual-Luciferase Reporter assay kit (Promega).

    Techniques: Expressing, Luciferase, Sequencing, Activity Assay

    IGF2BP1 modulates FN1 and SNAI2 (SLUG) transcription via LEF1. ( A ) Schematic of luciferase reporters comprising the full-length in silico predicted (FN-839) or 5′-truncated fragments of the human FN1 promoter. The proposed transcription start is indicated by +1 with a reported 5′-UTR of 266 nt. Putative LEF1-binding sites predicted by ‘PROMO’ are depicted as white boxes with labels ‘1-5’ in 5′-to-3′ direction. ( B ) The Firefly luciferase activity of indicated promoter fragments or empty pGL4 vector was monitored in HEK293 cells on transient co-transfection with RFP or LEF1 for 30 h. Firefly activities were normalized by Renilla activities [relative luciferase units (RLU)], serving as internal controls. All reporters comprising the putative LEF1-binding site four showed promoter activity and were activated by LEF1. ( C and D ) Binding of endogenous LEF1 protein to the human FN1 promoter in HEK293 cells was assessed by ChIP. The association of endogenous LEF1 or histone H3 to the FN1 promoter was monitored by semi-quantitative (C) as well as quantitative PCR (D) using to FN1 promoter specific amplicons (P1 and P2, indicated in lower panel). An intergenic probe served as positive control. IgG-agarose was used to monitor unspecific binding (C, negative control). In (D), the enrichment of indicated genomic DNA fragments (P1 and P2) or the intergenic control (intergenic) was determined relative to the diluted input fraction (I) normalized by IgG-controls using the ΔC t -method. ( E ) HEK293 cells were co-transfected with FN-839 luciferase reporter and IGF2BP1-directed (shI1-1), LEF1-directed (shL1-1) or control shRNA encoding vectors for 48 h. RLUs were determined as described in (B). ( F ) HEK293 cells were transfected with IGF2BP1-directed (siI1-2) or control siRNAs (siC) for 72 h. The abundance of SNAI2 mRNA in response to IGF2BP1 knockdown was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. ACTB served as control. ( G ) HEK293 cells transfected as in (F) were treated with ActD (5 µM) to block transcription for indicated times. SNAI2 mRNA turnover was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. RNA decay is depicted in semi-logarithmic scale revealing no significant difference in mRNA turnover ( P -value not shown). ( H ) HEK293 cells were transfected with LEF1-directed (siL1-1) or control siRNAs (siC) for 72 h. The abundance of SNAI2 mRNA in response to LEF1 depletion was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. RPLP0 served as control. ( I ) Schematic of Firefly luciferase reporters comprising the SNAI1 or SNAI2 promoter sequences, as previously reported ( 37 , 45 ). Indicated putative LEF1-binding sites within the SNAI1 or SNAI2 promoter were predicted [white boxes; as described in (A)] or as previously reported [gray boxes, only for SNAI2; ( 37 )]. ( J ) The Firefly activity of SNAI1 or SNAI2 promoter fragments cloned in pGL4 as well as the activity of empty pGL4 vector was monitored in HEK293 cells on transient co-transfection with RFP or LEF1 for 30 h. RLUs were determined as described in (B). LEF1 only enhanced the activity of the SNAI2 promoter. ( K ) HEK293 cells were co-transfected with SNAI1 or SNAI2 promoter reporters and indicated shRNA-encoding vectors for 48 h. RLUs were determined as described in (B). SNAI2 promoter activity was reduced by IGF2BP1 as well as LEF1 knockdown, whereas the SNAI1 reporter activity remained largely unaffected and was barely elevated compared with the empty control reporter. Statistical significance was validated by Student’s t -testing: * P

    Journal: Nucleic Acids Research

    Article Title: IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)

    doi: 10.1093/nar/gkt410

    Figure Lengend Snippet: IGF2BP1 modulates FN1 and SNAI2 (SLUG) transcription via LEF1. ( A ) Schematic of luciferase reporters comprising the full-length in silico predicted (FN-839) or 5′-truncated fragments of the human FN1 promoter. The proposed transcription start is indicated by +1 with a reported 5′-UTR of 266 nt. Putative LEF1-binding sites predicted by ‘PROMO’ are depicted as white boxes with labels ‘1-5’ in 5′-to-3′ direction. ( B ) The Firefly luciferase activity of indicated promoter fragments or empty pGL4 vector was monitored in HEK293 cells on transient co-transfection with RFP or LEF1 for 30 h. Firefly activities were normalized by Renilla activities [relative luciferase units (RLU)], serving as internal controls. All reporters comprising the putative LEF1-binding site four showed promoter activity and were activated by LEF1. ( C and D ) Binding of endogenous LEF1 protein to the human FN1 promoter in HEK293 cells was assessed by ChIP. The association of endogenous LEF1 or histone H3 to the FN1 promoter was monitored by semi-quantitative (C) as well as quantitative PCR (D) using to FN1 promoter specific amplicons (P1 and P2, indicated in lower panel). An intergenic probe served as positive control. IgG-agarose was used to monitor unspecific binding (C, negative control). In (D), the enrichment of indicated genomic DNA fragments (P1 and P2) or the intergenic control (intergenic) was determined relative to the diluted input fraction (I) normalized by IgG-controls using the ΔC t -method. ( E ) HEK293 cells were co-transfected with FN-839 luciferase reporter and IGF2BP1-directed (shI1-1), LEF1-directed (shL1-1) or control shRNA encoding vectors for 48 h. RLUs were determined as described in (B). ( F ) HEK293 cells were transfected with IGF2BP1-directed (siI1-2) or control siRNAs (siC) for 72 h. The abundance of SNAI2 mRNA in response to IGF2BP1 knockdown was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. ACTB served as control. ( G ) HEK293 cells transfected as in (F) were treated with ActD (5 µM) to block transcription for indicated times. SNAI2 mRNA turnover was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. RNA decay is depicted in semi-logarithmic scale revealing no significant difference in mRNA turnover ( P -value not shown). ( H ) HEK293 cells were transfected with LEF1-directed (siL1-1) or control siRNAs (siC) for 72 h. The abundance of SNAI2 mRNA in response to LEF1 depletion was analyzed by qRT-PCR using the ΔΔC t -method and PPIA for normalization. RPLP0 served as control. ( I ) Schematic of Firefly luciferase reporters comprising the SNAI1 or SNAI2 promoter sequences, as previously reported ( 37 , 45 ). Indicated putative LEF1-binding sites within the SNAI1 or SNAI2 promoter were predicted [white boxes; as described in (A)] or as previously reported [gray boxes, only for SNAI2; ( 37 )]. ( J ) The Firefly activity of SNAI1 or SNAI2 promoter fragments cloned in pGL4 as well as the activity of empty pGL4 vector was monitored in HEK293 cells on transient co-transfection with RFP or LEF1 for 30 h. RLUs were determined as described in (B). LEF1 only enhanced the activity of the SNAI2 promoter. ( K ) HEK293 cells were co-transfected with SNAI1 or SNAI2 promoter reporters and indicated shRNA-encoding vectors for 48 h. RLUs were determined as described in (B). SNAI2 promoter activity was reduced by IGF2BP1 as well as LEF1 knockdown, whereas the SNAI1 reporter activity remained largely unaffected and was barely elevated compared with the empty control reporter. Statistical significance was validated by Student’s t -testing: * P

    Article Snippet: Luciferase reporter analysis Luciferase activities were determined using DualGlo reagent (Promega), as previously reported ( , ).

    Techniques: Luciferase, In Silico, Binding Assay, Activity Assay, Plasmid Preparation, Cotransfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Transfection, shRNA, Quantitative RT-PCR, Blocking Assay, Clone Assay

    IGF2BP1 promotes LEF1 expression by preventing LEF1 mRNA degradation. ( A and B ) HEK293 cells were transfected with control (siC) or indicated IGF2BP1-directed (siI1-1, siI1-2) siRNAs for 72 h. Protein abundance on IGF2BP1 knockdown was determined relative to controls (siC) by western blotting using VCL and TUBA4A for cross-normalization, as indicated above panels. Representative western blots of three independent analyses are shown. ACTB and LEF1 mRNA levels were analyzed by qRT-PCR. Changes in RNA abundance on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) by the ΔΔC t -method using PPIA for normalization. ( C ) RNA decay was monitored in HEK293 cells transfected with indicated siRNAs for 72 h by blocking mRNA synthesis using ActD (5 µM) for indicated times. RNA levels were determined by qRT-PCR using normalization to PPIA by the ΔΔC t -method. RPLP0 served as a control. RNA decay is depicted in semi-logarithmic scale. Statistical significance determined over three independent analyses was analyzed by Student’s t -test, as shown in panels ( P -values). ( D and E ) The association of indicated mRNAs with IGF2BP1 in HEK293 cells was analyzed by RIP using formaldehyde fixation to stabilize mRNPs prior purification. Endogenous IGF2BP1 was immunopurified (I1) by a monoclonal antibody, as indicated by western blotting in the lower panel (IB). Co-purification of indicated mRNAs was analyzed relative to the input fraction (I, 10% of cell lysates) by semi-quantitative (D) as well as qRT-PCR (E). IgG-agarose served as a control (C) for unspecific mRNA binding. The enrichment of mRNAs by immunopurification of IGF2BP1 (I1) was determined relative to the input fraction by using the ΔC t -method (E). ( F ) Upper panel: Scheme of used Firefly reporters comprising the two alternative LEF1 3′-UTRs (A: Acc.No., NM_016269 /001130713/ 001166119; B: Acc.No., NM_001130714) or the vector-encoded BGH-3′UTR (C). Lower panel: HEK293 cells were transfected with control or indicated IGF2BP1-directed siRNAs for 48 h before the co-transfection of Firefly luciferase reporters (A–C: see scheme in upper panel) and Renilla luciferase control reporters for 24 h. Changes in Firefly luciferase reporter activities on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) on normalization by Renilla activities. Statistical significance was validated by Student’s t -test: * P

    Journal: Nucleic Acids Research

    Article Title: IGF2BP1 promotes mesenchymal cell properties and migration of tumor-derived cells by enhancing the expression of LEF1 and SNAI2 (SLUG)

    doi: 10.1093/nar/gkt410

    Figure Lengend Snippet: IGF2BP1 promotes LEF1 expression by preventing LEF1 mRNA degradation. ( A and B ) HEK293 cells were transfected with control (siC) or indicated IGF2BP1-directed (siI1-1, siI1-2) siRNAs for 72 h. Protein abundance on IGF2BP1 knockdown was determined relative to controls (siC) by western blotting using VCL and TUBA4A for cross-normalization, as indicated above panels. Representative western blots of three independent analyses are shown. ACTB and LEF1 mRNA levels were analyzed by qRT-PCR. Changes in RNA abundance on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) by the ΔΔC t -method using PPIA for normalization. ( C ) RNA decay was monitored in HEK293 cells transfected with indicated siRNAs for 72 h by blocking mRNA synthesis using ActD (5 µM) for indicated times. RNA levels were determined by qRT-PCR using normalization to PPIA by the ΔΔC t -method. RPLP0 served as a control. RNA decay is depicted in semi-logarithmic scale. Statistical significance determined over three independent analyses was analyzed by Student’s t -test, as shown in panels ( P -values). ( D and E ) The association of indicated mRNAs with IGF2BP1 in HEK293 cells was analyzed by RIP using formaldehyde fixation to stabilize mRNPs prior purification. Endogenous IGF2BP1 was immunopurified (I1) by a monoclonal antibody, as indicated by western blotting in the lower panel (IB). Co-purification of indicated mRNAs was analyzed relative to the input fraction (I, 10% of cell lysates) by semi-quantitative (D) as well as qRT-PCR (E). IgG-agarose served as a control (C) for unspecific mRNA binding. The enrichment of mRNAs by immunopurification of IGF2BP1 (I1) was determined relative to the input fraction by using the ΔC t -method (E). ( F ) Upper panel: Scheme of used Firefly reporters comprising the two alternative LEF1 3′-UTRs (A: Acc.No., NM_016269 /001130713/ 001166119; B: Acc.No., NM_001130714) or the vector-encoded BGH-3′UTR (C). Lower panel: HEK293 cells were transfected with control or indicated IGF2BP1-directed siRNAs for 48 h before the co-transfection of Firefly luciferase reporters (A–C: see scheme in upper panel) and Renilla luciferase control reporters for 24 h. Changes in Firefly luciferase reporter activities on IGF2BP1 knockdown (siIGF2BP1) were determined relative to controls (siC) on normalization by Renilla activities. Statistical significance was validated by Student’s t -test: * P

    Article Snippet: Luciferase reporter analysis Luciferase activities were determined using DualGlo reagent (Promega), as previously reported ( , ).

    Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Blocking Assay, Purification, Copurification, Binding Assay, Immu-Puri, Plasmid Preparation, Cotransfection, Luciferase

    PTGS in wild-type and hyl1 protoplasts. ( A ) Protoplasts from wild-type plants were transfected with H 2 O (mock-transfected), a 35S::LUC plasmid, and both the 35S::LUC and a LUC-dsRNA. After 12–16 h at 22°C, the transfected protoplasts were lysed, and LUC activity was measured (the lysis buffer was used as a control for the LUC assay). ( B ) The experiment was done by using hyl1 protoplasts transfected with both 35S::LUC and 35S::GUS genes; the protoplasts were then subdivided, and the indicated dsRNAs were added to aliquots, which were then incubated and assayed for both activities.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation

    doi: 10.1073/pnas.0307969100

    Figure Lengend Snippet: PTGS in wild-type and hyl1 protoplasts. ( A ) Protoplasts from wild-type plants were transfected with H 2 O (mock-transfected), a 35S::LUC plasmid, and both the 35S::LUC and a LUC-dsRNA. After 12–16 h at 22°C, the transfected protoplasts were lysed, and LUC activity was measured (the lysis buffer was used as a control for the LUC assay). ( B ) The experiment was done by using hyl1 protoplasts transfected with both 35S::LUC and 35S::GUS genes; the protoplasts were then subdivided, and the indicated dsRNAs were added to aliquots, which were then incubated and assayed for both activities.

    Article Snippet: The LUC and GUS activities were measured by using the LUC assay system (Promega) and the Aurora TM GUS chemiluminescent reporter gene assay (ICN), respectively, by using a Sirius luminometer (Berthold, Nashua, NH).

    Techniques: Transfection, Plasmid Preparation, Activity Assay, Lysis, Incubation

    Deletions define a 519-bp putative GPR43 promoter containing the core and proximal promoter. (a) ECR Browser plot spanning −4560 bp upstream of the transcription start site (+1), the entire GPR43 gene and 2000 bp after the gene. The plot represents sequence homology of the mouse chromosome 7 (Ref Seq ID: NC_000073.6 ) region flanking the murine Gpr43 (Entrez Gene ID: 233079) in comparison to the corresponding region on the human chromosome 19 (RefSeq ID: NC_000019.9; positions: 35934599–35942669), which is acting as the baseline. Sequence lengths of > 100 bp with 70% sequence conservation are shown as peaks. (b) Luciferase reporter activities of 5′ deletion constructs from a 4628 bp putative GPR43 promoter in differentiated U937 cells 22 h after transfection. Analysis of the homology with mouse sequence was used to approximate the deletion sites. Results represent the average Firefly luciferase read-outs of three independent transfections (n = 3) normalized to Renilla luciferase activity and relative to the basic (empty) luciferase vector, arbitrarily set as 1. Error bars represent the mean ± s.d.. The data shown are representative of three independent experiments. Two tailed Students' T-test was used to determine the statistical significance of the difference between promoter constructs and is annotated as: *

    Journal: Scientific Reports

    Article Title: The short-chain fatty acid receptor GPR43 is transcriptionally regulated by XBP1 in human monocytes

    doi: 10.1038/srep08134

    Figure Lengend Snippet: Deletions define a 519-bp putative GPR43 promoter containing the core and proximal promoter. (a) ECR Browser plot spanning −4560 bp upstream of the transcription start site (+1), the entire GPR43 gene and 2000 bp after the gene. The plot represents sequence homology of the mouse chromosome 7 (Ref Seq ID: NC_000073.6 ) region flanking the murine Gpr43 (Entrez Gene ID: 233079) in comparison to the corresponding region on the human chromosome 19 (RefSeq ID: NC_000019.9; positions: 35934599–35942669), which is acting as the baseline. Sequence lengths of > 100 bp with 70% sequence conservation are shown as peaks. (b) Luciferase reporter activities of 5′ deletion constructs from a 4628 bp putative GPR43 promoter in differentiated U937 cells 22 h after transfection. Analysis of the homology with mouse sequence was used to approximate the deletion sites. Results represent the average Firefly luciferase read-outs of three independent transfections (n = 3) normalized to Renilla luciferase activity and relative to the basic (empty) luciferase vector, arbitrarily set as 1. Error bars represent the mean ± s.d.. The data shown are representative of three independent experiments. Two tailed Students' T-test was used to determine the statistical significance of the difference between promoter constructs and is annotated as: *

    Article Snippet: Renilla luciferase reporter activities were assessed using the Dual Luciferase Reporter Assay System (Promega) 22 h after transfection.

    Techniques: Sequencing, Luciferase, Construct, Transfection, Activity Assay, Plasmid Preparation, Two Tailed Test

    Dual luciferase reporter assays. Relative luciferase activities measured from the SCG neurons ( a , c , and e ) or Neuro-2a cells ( b , d , and f ) overexpressing the indicated reporter constructs. The neurons (3–4 repeats on the independent cultures) were microinjected with the reporter plasmids together with the plasmid for eGFP. The ratio of Firefly and Renilla luciferase (Fluc/Rluc) activity was determined 48 h later and was normalized to the number of eGFP-positive neurons, counted before the lysis. The Neuro-2a cells (6 repeats on the independent cultures) were transiently transfected and the Fluc/Rluc activity determined 48 h later. Shown are the means±S.E.M. Statistical significance of the differences was estimated by Student's t -test ( a and b ) or one-way ANOVA and post hoc Tukey's test ( b and c ). * P

    Journal: Cell Death & Disease

    Article Title: Multiple mechanisms repress N-Bak mRNA translation in the healthy and apoptotic neurons

    doi: 10.1038/cddis.2013.297

    Figure Lengend Snippet: Dual luciferase reporter assays. Relative luciferase activities measured from the SCG neurons ( a , c , and e ) or Neuro-2a cells ( b , d , and f ) overexpressing the indicated reporter constructs. The neurons (3–4 repeats on the independent cultures) were microinjected with the reporter plasmids together with the plasmid for eGFP. The ratio of Firefly and Renilla luciferase (Fluc/Rluc) activity was determined 48 h later and was normalized to the number of eGFP-positive neurons, counted before the lysis. The Neuro-2a cells (6 repeats on the independent cultures) were transiently transfected and the Fluc/Rluc activity determined 48 h later. Shown are the means±S.E.M. Statistical significance of the differences was estimated by Student's t -test ( a and b ) or one-way ANOVA and post hoc Tukey's test ( b and c ). * P

    Article Snippet: Luciferase reporter assay Luciferase activity assay was performed using Dual-Luciferase Reporter Assay System (Promega) as published earlier.

    Techniques: Luciferase, Construct, Plasmid Preparation, Activity Assay, Lysis, Transfection

    The ectopic expression of HBx increases the activity and transcription of IGF2-P3 and P4 promoters in vitro. Huh-7 cells were transiently cotransfected with increasing amounts of HBx expression plasmid or control plasmid and premethylated pGL3-P3 or premethylated pGL3-P4 vectors. The observed firefly luciferase activity was normalized to Renilla luciferase activity and the results were the average of three independent experiments carried out in triplicate. The transfected quantities of HBx expression plasmid were 0.25 μg (+) and 0.5 μg (++) per 1 × 10 6 cells. *indicates that the mean values in Huh-7 cells cotransfected with control plasmid are significantly different from those cotransfected with HBx expression plasmid (P = 0.000).

    Journal: American Journal of Cancer Research

    Article Title: Epigenetic modulation of insulin-like growth factor-II overexpression by hepatitis B virus X protein in hepatocellular carcinoma

    doi:

    Figure Lengend Snippet: The ectopic expression of HBx increases the activity and transcription of IGF2-P3 and P4 promoters in vitro. Huh-7 cells were transiently cotransfected with increasing amounts of HBx expression plasmid or control plasmid and premethylated pGL3-P3 or premethylated pGL3-P4 vectors. The observed firefly luciferase activity was normalized to Renilla luciferase activity and the results were the average of three independent experiments carried out in triplicate. The transfected quantities of HBx expression plasmid were 0.25 μg (+) and 0.5 μg (++) per 1 × 10 6 cells. *indicates that the mean values in Huh-7 cells cotransfected with control plasmid are significantly different from those cotransfected with HBx expression plasmid (P = 0.000).

    Article Snippet: The pGL3-P3 and pGL3-P4 vectors were constructed by inserting the P3 promoter (-1251/+123) and the P4 promoter (-1129/+117), respectively, of the human IGF-II gene (GenBank Accession No. ) into the luciferase reporter vector pGL3-Basic (Promega, Madison, WI, USA).

    Techniques: Expressing, Activity Assay, In Vitro, Plasmid Preparation, Luciferase, Transfection