luciferase reporter assay Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • luc  (Promega)
    99
    Promega luc
    MDH and <t>Luc</t> cross-linking results mapped to the <t>PsHsp18.1</t> structure reveal that the main interaction regions are only fully exposed in the sHSP dimer. PsHsp18.1 Bpa variants protecting each substrate equivalently to wild-type were used for this analysis.
    Luc, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 10279 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luc/product/Promega
    Average 99 stars, based on 10279 article reviews
    Price from $9.99 to $1999.99
    luc - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Promega dual luciferase reporter assay system
    Subcellular localization and transcriptional regulation of CsGAPDHs proteins. (A, E, I, M) Analysis of the subcellular localization of the CsGAPDHs protein in tobacco leaf. The merged pictures (D, H, L, P) include the green fluorescence channel (first panels), the chloroplast autofluorescence channel (second panels) (B, F, J, N) and the bright field channel (third panels) (C, G, K, O). (Q) The main components of the vectors (effector, <t>reporter,</t> and the Internal control) are displayed. (R) Relative <t>luciferase</t> activity of CsGAPDH1 and CsGAPDH2 on promoters of the GAL4 using a <t>dual-luciferase</t> transient <t>assay</t> in Arabidopsis protoplasts with GAL4DB as control.
    Dual Luciferase Reporter Assay System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 142244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dual luciferase reporter assay system/product/Promega
    Average 99 stars, based on 142244 article reviews
    Price from $9.99 to $1999.99
    dual luciferase reporter assay system - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    92
    Promega dual luciferase reporter assay kit
    FHL2, up-regulated in OC tissues and cell lines, was a target of miR-195-5p. a , b The expression of FHL2 at mRNA level and protein level in OC tissues and adjacent healthy tissues was measured by qRT-PCR and western blot, respectively. c , d Spearman’s correlation analysis revealed the correlation between FHL2 mRNA level and miR-195-5p level or DLX6-AS1 level in OC tissues. e The expression of FHL2 in IOSE80, SKOV3 and A2780 was measured by western blot at the protein level. f Bioinformatics tool starBase analyzed the binding sites between FHL2 3′ UTR and miR-195-5p. g , h The relationship between FHL2 and miR-195-5p was confirmed by <t>dual-luciferase</t> <t>reporter</t> <t>assay.</t> i The interaction between FHL2 and miR-195-5p was further confirmed by RIP assay. j The protein level of FHL2 was detected by western blot in SKOV3 and A2780 cells transfected with miR-195-5p, NC, miR-195-5p + pcDNA-DLX6-AS1 or miR-195-5p + pcDNA-NC. * P
    Dual Luciferase Reporter Assay Kit, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 21377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dual luciferase reporter assay kit/product/Promega
    Average 92 stars, based on 21377 article reviews
    Price from $9.99 to $1999.99
    dual luciferase reporter assay kit - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    95
    Promega dual luciferase reporter assay
    MXDs require SID domains to activate and repress genes (A) DNA binding of MXDs does not require SID domains. ChIP-qPCR analysis of showing that DOX-induced (24 hours) WT and ΔSID versions of MNT, MXD1, and MXD2 bind to NCL and p21 genes in U2OStx cells (n=3). (B , C) The SID domains of MXDs are required for repression of 6xEbox-luc and activation of p21-luc. (B) <t>Luciferase</t> <t>reporter</t> <t>assay</t> of 6xEbox-luc in HEK293 cells transfected with wild type or ΔSID MNT, MXD1, and MXD2 (n=3). (C) p21-luc expression in HEK293 cells transfected with MIZ1 and co-transfected with wild type or ΔSID versions of MXDs (n=3). * P
    Dual Luciferase Reporter Assay, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 19826 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dual luciferase reporter assay/product/Promega
    Average 95 stars, based on 19826 article reviews
    Price from $9.99 to $1999.99
    dual luciferase reporter assay - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    93
    Promega pgl3 basic luciferase reporter vector
    DEDa leads to increased amount of active caspase-8 and sensitizes HeLa cells to TRAIL-induced apoptosis. ( A ) SH-SY5Y and A549 cells were separately cotransfected with <t>pGL3-C8(−470∼+76)</t> (1 μg) and an equal amount (0.5 μg)
    Pgl3 Basic Luciferase Reporter Vector, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 3436 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgl3 basic luciferase reporter vector/product/Promega
    Average 93 stars, based on 3436 article reviews
    Price from $9.99 to $1999.99
    pgl3 basic luciferase reporter vector - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    85
    Promega dual luc kit
    Effect of PXR activators on NF-κB-dependent gene expression in <t>U397-3XκB-LUC</t> cells. (A) Left panel, RT-PCR for PXR and GAPDH in <t>U937-3XκB-LUC</t> cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).
    Dual Luc Kit, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dual luc kit/product/Promega
    Average 85 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    dual luc kit - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    94
    Thermo Fisher pmir report luciferase vector
    miR-135b targets GSK3β. (A) Schematic of predicted miR-135b site in the 3′UTR of GSK3β mRNA, which broadly conserved among vertebrates. (B) GSK3β expression was much lower in U87R cells compared to U87 cells at protein level and mRNA level by qRT-PCR and Western blot. (C) GSK3β expression was much lower in U87R cells compared to U87 cells at protein level by immunofluorescence. (D) miR-135b expression interference negatively regulated GSK3β protein expression by Western blot. (E) miR-135b expression interference negatively regulated GSK3β protein expression by immunofluorescence. (F) Luciferase reporter assays were performed after transfection with indicated <t>pMIR-Report</t> plasmids and a renilla transfection control plasmid, co-transfected with miR-135, or relevant scramble controls. Data shown were the mean±SD of six replicates and were representative of three independent experiments. *p
    Pmir Report Luciferase Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmir report luciferase vector/product/Thermo Fisher
    Average 94 stars, based on 3112 article reviews
    Price from $9.99 to $1999.99
    pmir report luciferase vector - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    94
    Berthold Technologies luciferase activity
    miR-135b targets GSK3β. (A) Schematic of predicted miR-135b site in the 3′UTR of GSK3β mRNA, which broadly conserved among vertebrates. (B) GSK3β expression was much lower in U87R cells compared to U87 cells at protein level and mRNA level by qRT-PCR and Western blot. (C) GSK3β expression was much lower in U87R cells compared to U87 cells at protein level by immunofluorescence. (D) miR-135b expression interference negatively regulated GSK3β protein expression by Western blot. (E) miR-135b expression interference negatively regulated GSK3β protein expression by immunofluorescence. (F) Luciferase reporter assays were performed after transfection with indicated <t>pMIR-Report</t> plasmids and a renilla transfection control plasmid, co-transfected with miR-135, or relevant scramble controls. Data shown were the mean±SD of six replicates and were representative of three independent experiments. *p
    Luciferase Activity, supplied by Berthold Technologies, used in various techniques. Bioz Stars score: 94/100, based on 539 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luciferase activity/product/Berthold Technologies
    Average 94 stars, based on 539 article reviews
    Price from $9.99 to $1999.99
    luciferase activity - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    Image Search Results


    MDH and Luc cross-linking results mapped to the PsHsp18.1 structure reveal that the main interaction regions are only fully exposed in the sHSP dimer. PsHsp18.1 Bpa variants protecting each substrate equivalently to wild-type were used for this analysis.

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

    Article Title: Substrate binding site flexibility of the small heat shock protein molecular chaperones

    doi: 10.1073/pnas.0902177106

    Figure Lengend Snippet: MDH and Luc cross-linking results mapped to the PsHsp18.1 structure reveal that the main interaction regions are only fully exposed in the sHSP dimer. PsHsp18.1 Bpa variants protecting each substrate equivalently to wild-type were used for this analysis.

    Article Snippet: To determine chaperone efficiency of PsHsp18.1 Bpa variants, 2 μM of Luc (recombinant Photinus pyralis luciferase, 62-kDa monomer from Promega) or 6 μM pig heart mitochondrial MDH, 33-kDa monomer (active form is dimeric) (Roche) were incubated at 8.5 min at 42 °C or 120 min at 45 °C, respectively, with the indicated ratios of sHSP.

    Techniques:

    Substrate cross-linking to PsHsp18.1 reveals major interactions with the N-terminal arm. Comparison of 19 Bpa variants which protected both MDH and Luc at wild-type molar ratios of sHSP:substrate. Intensity of cross-linked species is shown as a percentage

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

    Article Title: Substrate binding site flexibility of the small heat shock protein molecular chaperones

    doi: 10.1073/pnas.0902177106

    Figure Lengend Snippet: Substrate cross-linking to PsHsp18.1 reveals major interactions with the N-terminal arm. Comparison of 19 Bpa variants which protected both MDH and Luc at wild-type molar ratios of sHSP:substrate. Intensity of cross-linked species is shown as a percentage

    Article Snippet: To determine chaperone efficiency of PsHsp18.1 Bpa variants, 2 μM of Luc (recombinant Photinus pyralis luciferase, 62-kDa monomer from Promega) or 6 μM pig heart mitochondrial MDH, 33-kDa monomer (active form is dimeric) (Roche) were incubated at 8.5 min at 42 °C or 120 min at 45 °C, respectively, with the indicated ratios of sHSP.

    Techniques:

    PsHsp18.1 cross-links to MDH and Luc only under substrate denaturing conditions. The indicated Bpa variant or wildtype PsHsp18.1 were heated (+, odd numbered lanes) or not (−, even numbered lanes) in the presence of Luc ( A ) or MDH ( B ). After UV-cross-linking

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

    Article Title: Substrate binding site flexibility of the small heat shock protein molecular chaperones

    doi: 10.1073/pnas.0902177106

    Figure Lengend Snippet: PsHsp18.1 cross-links to MDH and Luc only under substrate denaturing conditions. The indicated Bpa variant or wildtype PsHsp18.1 were heated (+, odd numbered lanes) or not (−, even numbered lanes) in the presence of Luc ( A ) or MDH ( B ). After UV-cross-linking

    Article Snippet: To determine chaperone efficiency of PsHsp18.1 Bpa variants, 2 μM of Luc (recombinant Photinus pyralis luciferase, 62-kDa monomer from Promega) or 6 μM pig heart mitochondrial MDH, 33-kDa monomer (active form is dimeric) (Roche) were incubated at 8.5 min at 42 °C or 120 min at 45 °C, respectively, with the indicated ratios of sHSP.

    Techniques: Variant Assay

    Some cellular interactors of nsP2 contribute to CHIKV replication in vitro . (A) Human HEK-293T cells were transfected with targeted siRNA to knock down cellular partners of nsP2 and infected 48 h later with a recombinant CHIKV expressing Renilla luciferase

    Journal: Journal of Virology

    Article Title: Mapping of Chikungunya Virus Interactions with Host Proteins Identified nsP2 as a Highly Connected Viral Component

    doi: 10.1128/JVI.06390-11

    Figure Lengend Snippet: Some cellular interactors of nsP2 contribute to CHIKV replication in vitro . (A) Human HEK-293T cells were transfected with targeted siRNA to knock down cellular partners of nsP2 and infected 48 h later with a recombinant CHIKV expressing Renilla luciferase

    Article Snippet: After 24 h of culture, Renilla luciferase activity was determined using the Renilla -Glo reagent (Promega) following the manufacturer's recommendations.

    Techniques: In Vitro, Transfection, Infection, Recombinant, Expressing, Luciferase

    PAK5-mediated phosphorylation promotes EMT in cervical cancer cells. a , b Effects of SATB1 and SATB1 Ser47A on the migration and invasion ability in HeLa and SiHa cells. Scale bar, 100μm for a and b. c HeLa and SiHa cells were transfected with pGL3-E, together with the indicated expression plasmids for Dual luciferase reporter assays. d Effects of SATB1 knockdown on EMT markers were analyzed by western blotting in HeLa cells overexpressing PAK5. e Effects of SATB1 overexpression on EMT markers in HeLa cells silencing PAK5. Data are showed as mean ± SD for three independent experiments. **, P

    Journal: Cell Death and Differentiation

    Article Title: PAK5 promotes the migration and invasion of cervical cancer cells by phosphorylating SATB1

    doi: 10.1038/s41418-018-0178-4

    Figure Lengend Snippet: PAK5-mediated phosphorylation promotes EMT in cervical cancer cells. a , b Effects of SATB1 and SATB1 Ser47A on the migration and invasion ability in HeLa and SiHa cells. Scale bar, 100μm for a and b. c HeLa and SiHa cells were transfected with pGL3-E, together with the indicated expression plasmids for Dual luciferase reporter assays. d Effects of SATB1 knockdown on EMT markers were analyzed by western blotting in HeLa cells overexpressing PAK5. e Effects of SATB1 overexpression on EMT markers in HeLa cells silencing PAK5. Data are showed as mean ± SD for three independent experiments. **, P

    Article Snippet: After 24 h transfection, cell lysates were harvested, and then luciferase activities were measured by the Dual-Luciferase Reporter System (Promega, Fitchburg, WI, USA) on a luminometer (Lumat LB 9507, EG & G Berthold, Bad Wildbad, Germany).

    Techniques: Migration, Transfection, Expressing, Luciferase, Western Blot, Over Expression

    miR-342 and miR-363 suppress Runx2 expression and the proliferation and migration of MM cells in vitro (A, B) Relative expression of Runx2 in CAG cells transfected with NS-miR-control or miR-342, miR-363, or miR-342+363 mimics was assessed by Western blotting (A) and RT-qPCR (B). (C, D) Non-specific (NS) miR-control or miR-342 (C) or miR-363 mimics (D) were co-transfected with either WT or mutant Runx2 3′UTR-Luc reporter plasmids into HEK293 cells (MT: mutant). Firefly Luc activity was normalized to co-transfected Renilla Luc activity and presented in relative luminescence units. (E) Cell viability was measured by MTT assay in CAG cells transfected with miR-Scramble control or miR-342, miR-363, or miR-342+363 mimics. (F) Transwell migration assays were conducted to assess cell migration in CAG cells transfected with miR-Scramble control or miR-342, miR-363, or miR-342+363 mimics. The P values were obtained by one-way ANOVA followed by Tukey-Kramer post hoc test (ns: non-significant; * P

    Journal: Molecular cancer research : MCR

    Article Title: Runx2 Suppression by miR-342 and miR-363 Inhibits Multiple Myeloma Progression

    doi: 10.1158/1541-7786.MCR-17-0606

    Figure Lengend Snippet: miR-342 and miR-363 suppress Runx2 expression and the proliferation and migration of MM cells in vitro (A, B) Relative expression of Runx2 in CAG cells transfected with NS-miR-control or miR-342, miR-363, or miR-342+363 mimics was assessed by Western blotting (A) and RT-qPCR (B). (C, D) Non-specific (NS) miR-control or miR-342 (C) or miR-363 mimics (D) were co-transfected with either WT or mutant Runx2 3′UTR-Luc reporter plasmids into HEK293 cells (MT: mutant). Firefly Luc activity was normalized to co-transfected Renilla Luc activity and presented in relative luminescence units. (E) Cell viability was measured by MTT assay in CAG cells transfected with miR-Scramble control or miR-342, miR-363, or miR-342+363 mimics. (F) Transwell migration assays were conducted to assess cell migration in CAG cells transfected with miR-Scramble control or miR-342, miR-363, or miR-342+363 mimics. The P values were obtained by one-way ANOVA followed by Tukey-Kramer post hoc test (ns: non-significant; * P

    Article Snippet: Luc assays were performed with HEK293 cells co-transfected with the specified miRNA mimics (100 nM each) or NS control miRNA (100 nM) and the specified Runx2 3′UTR-Luc reporter (200 ng) plasmid using 6 μL of Fugene transfection reagent (Promega, Madison, WI) in triplicate in six independent experiments.

    Techniques: Expressing, Migration, In Vitro, Transfection, Western Blot, Quantitative RT-PCR, Mutagenesis, Activity Assay, MTT 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

    RNA polymerase III transcription can be measured in vivo using a RNA pol III dual luciferase assay. (A) Transient transfection of HeLa cells with increasing concentrations of pGL3-U6 (100ng, 200ng, 300ng), pGL3-VAI (100ng, 200ng, 300ng) or empty pGL3 vector (300ng). (B) Luciferase activity comparison of the wild-type human U6 promoter (100ng) with a mutant human U6 promoter, lacking a functional TATA box and PSE (100ng). Cells were also transfected with empty pGL3 (100ng) as negative control. (C) Comparison of wild-type VAI (100ng) promoter luciferase activity with a mutant VAI construct lacking both the A and B box promoter elements of the VAI promoter (100ng). Cells were also transfected with empty pGL3 (100ng) as negative control. (D) HeLa cells transiently transfected with either empty pGL3 (50ng), wild-type pGL3-U6 (50ng), or a previously characterized mutant U6 lacking a functional TATA box and transcribed by RNA pol II, pGL3-mtTATAU6 (25ng) and treated with 0 μg/ml or 3 μg/ml α-amanitin. (E) HeLa cells transiently transfected with either empty pGL3 (100ng), wild-type pGL3-VAI (100ng), or a previously characterized mutant U6 lacking a functional TATA box and transcribed by RNA pol II, pGL3-mtTATAU6 (50ng) and treated with 0 μg/ml, 1 μg/ml or 2 μg/ml α-amanitin. (F) Transient transfection of pGL3 (100ng), pGL3-U6 (100ng), or pGL3-U6TTT (100ng) with a run of T residues inserted downstream of the human U6 promoter, but upstream of the the Kozak consensus translation initiation site of the luciferase gene in pGL3. All luciferase assay results expressed as relative light units (RLU): the average of the Photinus pyralis firefly activity observed divided by the average of the activity recorded from Renilla luciferase vector. Experiments were done in triplicate, repeated three times, representative experiments are depicted.

    Journal: International Journal of Biological Sciences

    Article Title: Human Maf1 negatively regulates RNA Polymerase III transcription via the TFIIB family members Brf1 and Brf2

    doi:

    Figure Lengend Snippet: RNA polymerase III transcription can be measured in vivo using a RNA pol III dual luciferase assay. (A) Transient transfection of HeLa cells with increasing concentrations of pGL3-U6 (100ng, 200ng, 300ng), pGL3-VAI (100ng, 200ng, 300ng) or empty pGL3 vector (300ng). (B) Luciferase activity comparison of the wild-type human U6 promoter (100ng) with a mutant human U6 promoter, lacking a functional TATA box and PSE (100ng). Cells were also transfected with empty pGL3 (100ng) as negative control. (C) Comparison of wild-type VAI (100ng) promoter luciferase activity with a mutant VAI construct lacking both the A and B box promoter elements of the VAI promoter (100ng). Cells were also transfected with empty pGL3 (100ng) as negative control. (D) HeLa cells transiently transfected with either empty pGL3 (50ng), wild-type pGL3-U6 (50ng), or a previously characterized mutant U6 lacking a functional TATA box and transcribed by RNA pol II, pGL3-mtTATAU6 (25ng) and treated with 0 μg/ml or 3 μg/ml α-amanitin. (E) HeLa cells transiently transfected with either empty pGL3 (100ng), wild-type pGL3-VAI (100ng), or a previously characterized mutant U6 lacking a functional TATA box and transcribed by RNA pol II, pGL3-mtTATAU6 (50ng) and treated with 0 μg/ml, 1 μg/ml or 2 μg/ml α-amanitin. (F) Transient transfection of pGL3 (100ng), pGL3-U6 (100ng), or pGL3-U6TTT (100ng) with a run of T residues inserted downstream of the human U6 promoter, but upstream of the the Kozak consensus translation initiation site of the luciferase gene in pGL3. All luciferase assay results expressed as relative light units (RLU): the average of the Photinus pyralis firefly activity observed divided by the average of the activity recorded from Renilla luciferase vector. Experiments were done in triplicate, repeated three times, representative experiments are depicted.

    Article Snippet: All luciferase results are reported as relative light units (RLU): the average of the Photinus pyralis firefly activity observed divided by the average of the activity recorded from Renilla luciferase vector.

    Techniques: In Vivo, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Mutagenesis, Functional Assay, Negative Control, Construct

    LXRα co-activates ChREBP specific target genes in vitro and in vivo . A. Huh7 cells cultured in 25 mM glucose were transfected with a Chrebpβ (n=3) or Lpk -driven luciferase reporter (n=6), and plasmids expressing ChREBPα/Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Six hours post transfection, cells were treated with 2.5 mM or 25 mM glucose for 18 hours. Dual luciferase reporter assays were performed 24 hours post transfection. B. LXRαβ wild type (WT), LXRα -/- , LXRβ -/- and LXRα -/- β -/- mice were fasted for 24 hours (white bars) or fasted for 24 hours and refed for 12 hours (black bars) (n=5-8 mice per group). Hepatic gene expression of Lpk ( Pklr ), Chrebpβ and Chrebpα was analyzed by quantitative RT-PCR and normalized to Tbp . Data are presented as mean ± standard error of the mean (SEM). Significant differences are shown as *p

    Journal: bioRxiv

    Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

    doi: 10.1101/2019.12.20.869974

    Figure Lengend Snippet: LXRα co-activates ChREBP specific target genes in vitro and in vivo . A. Huh7 cells cultured in 25 mM glucose were transfected with a Chrebpβ (n=3) or Lpk -driven luciferase reporter (n=6), and plasmids expressing ChREBPα/Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Six hours post transfection, cells were treated with 2.5 mM or 25 mM glucose for 18 hours. Dual luciferase reporter assays were performed 24 hours post transfection. B. LXRαβ wild type (WT), LXRα -/- , LXRβ -/- and LXRα -/- β -/- mice were fasted for 24 hours (white bars) or fasted for 24 hours and refed for 12 hours (black bars) (n=5-8 mice per group). Hepatic gene expression of Lpk ( Pklr ), Chrebpβ and Chrebpα was analyzed by quantitative RT-PCR and normalized to Tbp . Data are presented as mean ± standard error of the mean (SEM). Significant differences are shown as *p

    Article Snippet: Dual-Luciferase® Reporter Assays (Promega, #E1960) were run on a Synergy H1 plate reader (BioTek® Instruments, Winooski, VT) according to manufacturer’s manual.

    Techniques: In Vitro, In Vivo, Cell Culture, Transfection, Luciferase, Expressing, Mouse Assay, Quantitative RT-PCR

    LXRα:ChREBPα co-activation requires functional ChoREs but not LXREs. A. Schematic representation of the synthetic luciferase reporters constructs. ChoRE, carbohydrate response element; LXRE, LXR response element. B. Huh7 cells cultured in 25 mM glucose were transfected with synthetic luciferase reporters containing ChoRE+LXRE, ChoRE-only, or LXRE-only, and plasmids expressing ChREBPα, the activated quadruple mutant ChREBP-Q, or ChREBPβ, together with Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Dual luciferase reporter assays were performed 24 hours post transfection. C. Huh7 cells cultured in 25 mM glucose were transfected with a Chrebpβ or Lpk -driven luciferase reporter luciferase reporter, and plasmids expressing ChREBPα, ChREBP-Q, ChREBPβ together with Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Dual luciferase reporter assays were performed 24 hours post transfection. Data are presented as mean ± SEM (n=3-7). Significant differences are shown as *p

    Journal: bioRxiv

    Article Title: Open the LID: LXRα regulates ChREBPα transactivity in a target gene-specific manner through an agonist-modulated LBD-LID interaction

    doi: 10.1101/2019.12.20.869974

    Figure Lengend Snippet: LXRα:ChREBPα co-activation requires functional ChoREs but not LXREs. A. Schematic representation of the synthetic luciferase reporters constructs. ChoRE, carbohydrate response element; LXRE, LXR response element. B. Huh7 cells cultured in 25 mM glucose were transfected with synthetic luciferase reporters containing ChoRE+LXRE, ChoRE-only, or LXRE-only, and plasmids expressing ChREBPα, the activated quadruple mutant ChREBP-Q, or ChREBPβ, together with Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Dual luciferase reporter assays were performed 24 hours post transfection. C. Huh7 cells cultured in 25 mM glucose were transfected with a Chrebpβ or Lpk -driven luciferase reporter luciferase reporter, and plasmids expressing ChREBPα, ChREBP-Q, ChREBPβ together with Mlxγ, with or without LXRα/RXRα. The Renilla luciferase reporter pRL-CMV was used as internal control. Dual luciferase reporter assays were performed 24 hours post transfection. Data are presented as mean ± SEM (n=3-7). Significant differences are shown as *p

    Article Snippet: Dual-Luciferase® Reporter Assays (Promega, #E1960) were run on a Synergy H1 plate reader (BioTek® Instruments, Winooski, VT) according to manufacturer’s manual.

    Techniques: Activation Assay, Functional Assay, Luciferase, Construct, Cell Culture, Transfection, Expressing, Mutagenesis

    Activation of PPARγ by apoptotic cells. RAW264.7 macrophages were transfected with the plasmids encoding ACOx-PPRE luciferase and Renilla luciferase and subsequently stimulated for 15 h with apoptotic Jurkat cells (AC) or THP-1 cells (AC T) generated

    Journal: Journal of Lipid Research

    Article Title: Attenuated suppression of the oxidative burst by cells dying in the presence of oxidized low density lipoprotein [S]

    doi: 10.1194/jlr.M800615-JLR200

    Figure Lengend Snippet: Activation of PPARγ by apoptotic cells. RAW264.7 macrophages were transfected with the plasmids encoding ACOx-PPRE luciferase and Renilla luciferase and subsequently stimulated for 15 h with apoptotic Jurkat cells (AC) or THP-1 cells (AC T) generated

    Article Snippet: On the next day, cells were treated with 2 × 106 apoptotic cells for 15 h prior to analysis of firefly and Renilla luciferase activities (dual luciferase reporter assay system, Promega).

    Techniques: Activation Assay, Transfection, Luciferase, Generated

    Subcellular localization and transcriptional regulation of CsGAPDHs proteins. (A, E, I, M) Analysis of the subcellular localization of the CsGAPDHs protein in tobacco leaf. The merged pictures (D, H, L, P) include the green fluorescence channel (first panels), the chloroplast autofluorescence channel (second panels) (B, F, J, N) and the bright field channel (third panels) (C, G, K, O). (Q) The main components of the vectors (effector, reporter, and the Internal control) are displayed. (R) Relative luciferase activity of CsGAPDH1 and CsGAPDH2 on promoters of the GAL4 using a dual-luciferase transient assay in Arabidopsis protoplasts with GAL4DB as control.

    Journal: PeerJ

    Article Title: Genome-wide identification, characterization, interaction network and expression profile of GAPDH gene family in sweet orange (Citrus sinensis)

    doi: 10.7717/peerj.7934

    Figure Lengend Snippet: Subcellular localization and transcriptional regulation of CsGAPDHs proteins. (A, E, I, M) Analysis of the subcellular localization of the CsGAPDHs protein in tobacco leaf. The merged pictures (D, H, L, P) include the green fluorescence channel (first panels), the chloroplast autofluorescence channel (second panels) (B, F, J, N) and the bright field channel (third panels) (C, G, K, O). (Q) The main components of the vectors (effector, reporter, and the Internal control) are displayed. (R) Relative luciferase activity of CsGAPDH1 and CsGAPDH2 on promoters of the GAL4 using a dual-luciferase transient assay in Arabidopsis protoplasts with GAL4DB as control.

    Article Snippet: The luciferase activities were measured with the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA).

    Techniques: Fluorescence, Luciferase, Activity Assay

    The targeting relationship between Axin1 and miR-128-3p. ( A ) The binding sites between miR-128-3p and Axin1 3'UTR predicted by Targetscan ( http://www.targetscan.org/vert_71/ ). ( B ) The targeting relationship between miR-128-3p and Axin1 verified using dual-luciferase reporter gene assay. In the X-axis, 3'UTR- Axin1 -WT refers to Axin1 wild type 3’UTR and 3'UTR- Axin1 -MUT refers to Axin1 mutant 3’UTR. * p

    Journal: Aging (Albany NY)

    Article Title: HIF-1α/microRNA-128-3p axis protects hippocampal neurons from apoptosis via the Axin1-mediated Wnt/β-catenin signaling pathway in Parkinson’s disease models

    doi: 10.18632/aging.102636

    Figure Lengend Snippet: The targeting relationship between Axin1 and miR-128-3p. ( A ) The binding sites between miR-128-3p and Axin1 3'UTR predicted by Targetscan ( http://www.targetscan.org/vert_71/ ). ( B ) The targeting relationship between miR-128-3p and Axin1 verified using dual-luciferase reporter gene assay. In the X-axis, 3'UTR- Axin1 -WT refers to Axin1 wild type 3’UTR and 3'UTR- Axin1 -MUT refers to Axin1 mutant 3’UTR. * p

    Article Snippet: The luciferase activity was measured using the Dual-Luciferase Reporter Assay System kit (Promega Corporation, Madison, WI, USA) on the fluorescence detector (Promega Corporation, Madison, WI, USA).

    Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Mutagenesis

    Evaluation of the NanoLuc reporter system by expressing murine NFAT1. ( A ) HEK293 cells were co-transfected with an expression vector of murine NFAT1, a firefly luciferase (Fluc) expression plasmid (pGL4.53[ luc2/ PGK]), and pNL3.2[ NlucP/ minP] containing different numbers (none, 3, 6 and 9) of the IL8 NFAT-RE. One day after transfection, cells were stimulated with ionomycin (IM, 1 μM; vehicle, 0.007% ethanol) for 6 h. Cell lysates were used to measure luminescent signals of NanoLuc (Nluc) and Fluc using a Nano-Glo Dual-Luciferase Reporter Assay System. The ratio of Nluc to Fluc, Nluc/Fluc, is expressed as normalized relative luciferase activity (RLA). Dots and bars represent individual and averaged RLA values obtained from triplicate assays, respectively. ( B ) HEK293 cells were co-transfected with expression plasmids for NanoLuc reporter and Fluc together with either wild type (WT), constitutively active type (CA) murine NFAT1 or empty vector (pcDNA3). One day after transfection, cells were pre-treated with FK506 (10 μM) or vehicle (0.16% ethanol) for 1 h and then subjected to ionomycin (IM) stimulation for 6 h, followed by luciferase assays.

    Journal: International Journal of Molecular Sciences

    Article Title: Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization

    doi: 10.3390/ijms19020605

    Figure Lengend Snippet: Evaluation of the NanoLuc reporter system by expressing murine NFAT1. ( A ) HEK293 cells were co-transfected with an expression vector of murine NFAT1, a firefly luciferase (Fluc) expression plasmid (pGL4.53[ luc2/ PGK]), and pNL3.2[ NlucP/ minP] containing different numbers (none, 3, 6 and 9) of the IL8 NFAT-RE. One day after transfection, cells were stimulated with ionomycin (IM, 1 μM; vehicle, 0.007% ethanol) for 6 h. Cell lysates were used to measure luminescent signals of NanoLuc (Nluc) and Fluc using a Nano-Glo Dual-Luciferase Reporter Assay System. The ratio of Nluc to Fluc, Nluc/Fluc, is expressed as normalized relative luciferase activity (RLA). Dots and bars represent individual and averaged RLA values obtained from triplicate assays, respectively. ( B ) HEK293 cells were co-transfected with expression plasmids for NanoLuc reporter and Fluc together with either wild type (WT), constitutively active type (CA) murine NFAT1 or empty vector (pcDNA3). One day after transfection, cells were pre-treated with FK506 (10 μM) or vehicle (0.16% ethanol) for 1 h and then subjected to ionomycin (IM) stimulation for 6 h, followed by luciferase assays.

    Article Snippet: Cell lysates were used to measure luminescent signals of Nluc and Fluc using a Nano-Glo® Dual-Luciferase Reporter Assay System provided from Promega (Fitchburg, WI, USA).

    Techniques: Expressing, Transfection, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay

    FHL2, up-regulated in OC tissues and cell lines, was a target of miR-195-5p. a , b The expression of FHL2 at mRNA level and protein level in OC tissues and adjacent healthy tissues was measured by qRT-PCR and western blot, respectively. c , d Spearman’s correlation analysis revealed the correlation between FHL2 mRNA level and miR-195-5p level or DLX6-AS1 level in OC tissues. e The expression of FHL2 in IOSE80, SKOV3 and A2780 was measured by western blot at the protein level. f Bioinformatics tool starBase analyzed the binding sites between FHL2 3′ UTR and miR-195-5p. g , h The relationship between FHL2 and miR-195-5p was confirmed by dual-luciferase reporter assay. i The interaction between FHL2 and miR-195-5p was further confirmed by RIP assay. j The protein level of FHL2 was detected by western blot in SKOV3 and A2780 cells transfected with miR-195-5p, NC, miR-195-5p + pcDNA-DLX6-AS1 or miR-195-5p + pcDNA-NC. * P

    Journal: Cancer Cell International

    Article Title: LncRNA DLX6-AS1 aggravates the development of ovarian cancer via modulating FHL2 by sponging miR-195-5p

    doi: 10.1186/s12935-020-01452-z

    Figure Lengend Snippet: FHL2, up-regulated in OC tissues and cell lines, was a target of miR-195-5p. a , b The expression of FHL2 at mRNA level and protein level in OC tissues and adjacent healthy tissues was measured by qRT-PCR and western blot, respectively. c , d Spearman’s correlation analysis revealed the correlation between FHL2 mRNA level and miR-195-5p level or DLX6-AS1 level in OC tissues. e The expression of FHL2 in IOSE80, SKOV3 and A2780 was measured by western blot at the protein level. f Bioinformatics tool starBase analyzed the binding sites between FHL2 3′ UTR and miR-195-5p. g , h The relationship between FHL2 and miR-195-5p was confirmed by dual-luciferase reporter assay. i The interaction between FHL2 and miR-195-5p was further confirmed by RIP assay. j The protein level of FHL2 was detected by western blot in SKOV3 and A2780 cells transfected with miR-195-5p, NC, miR-195-5p + pcDNA-DLX6-AS1 or miR-195-5p + pcDNA-NC. * P

    Article Snippet: After transfection for 48 h, the luciferase activity was observed using the Dual-Luciferase Reporter Assay Kit (Promega) by a Modulus Microplate Luminometer (Turner BioSystems Inc., Sunnyvale, CA, USA).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Binding Assay, Luciferase, Reporter Assay, Transfection

    MiR-195-5p was a target of DLX6-AS1 and its expression was declined in OC tissues and cell lines. a The expression of miR-195-5p in OC tissues and adjacent healthy tissues was measured by qRT-PCR. b Spearman’s correlation analysis revealed the correlation between DLX6-AS1 level and miR-195-5p level in OC tissues. c The expression of miR-195-5p in IOSE80, SKOV3 and A2780 was measured by qRT-PCR. d Bioinformatics tool starBase analyzed the binding sites between DLX6-AS1 and miR-195-5p. e , f The relationship between DLX6-AS1 and miR-195-5p was confirmed by dual-luciferase reporter assay. g The interaction between DLX6-AS1 and miR-195-5p was further confirmed by RIP assay. (H) The change of miR-195-5p expression level was detected by qRT-PCR in SKOV3 and A2780 cells with DLX6-AS1 overexpression or knockdown. * P

    Journal: Cancer Cell International

    Article Title: LncRNA DLX6-AS1 aggravates the development of ovarian cancer via modulating FHL2 by sponging miR-195-5p

    doi: 10.1186/s12935-020-01452-z

    Figure Lengend Snippet: MiR-195-5p was a target of DLX6-AS1 and its expression was declined in OC tissues and cell lines. a The expression of miR-195-5p in OC tissues and adjacent healthy tissues was measured by qRT-PCR. b Spearman’s correlation analysis revealed the correlation between DLX6-AS1 level and miR-195-5p level in OC tissues. c The expression of miR-195-5p in IOSE80, SKOV3 and A2780 was measured by qRT-PCR. d Bioinformatics tool starBase analyzed the binding sites between DLX6-AS1 and miR-195-5p. e , f The relationship between DLX6-AS1 and miR-195-5p was confirmed by dual-luciferase reporter assay. g The interaction between DLX6-AS1 and miR-195-5p was further confirmed by RIP assay. (H) The change of miR-195-5p expression level was detected by qRT-PCR in SKOV3 and A2780 cells with DLX6-AS1 overexpression or knockdown. * P

    Article Snippet: After transfection for 48 h, the luciferase activity was observed using the Dual-Luciferase Reporter Assay Kit (Promega) by a Modulus Microplate Luminometer (Turner BioSystems Inc., Sunnyvale, CA, USA).

    Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Reporter Assay, Over Expression

    The association between circNRIP1 and miR-182 in gastric cancer cells. ( A ) starBase online predicted the binding sites of circNRIP1 and miR-182. ( B – E ) Luciferase reporter assay and RIP were used to analyze the association between circNRIP1 and miR-182 in MGC-803 and AGS cells transfected with miR-182 or miR-NC. ( F and G ) qRT-PCR assay detected the abundance of miR-182 in MGC-803 and AGS cells transfected with pcDNA, circNRIP1, si-NC or si-circNRIP1. ** P

    Journal: OncoTargets and therapy

    Article Title: Down-Regulation of circNRIP1 Promotes the Apoptosis and Inhibits the Migration and Invasion of Gastric Cancer Cells by miR-182/ROCK1 Axis

    doi: 10.2147/OTT.S221633

    Figure Lengend Snippet: The association between circNRIP1 and miR-182 in gastric cancer cells. ( A ) starBase online predicted the binding sites of circNRIP1 and miR-182. ( B – E ) Luciferase reporter assay and RIP were used to analyze the association between circNRIP1 and miR-182 in MGC-803 and AGS cells transfected with miR-182 or miR-NC. ( F and G ) qRT-PCR assay detected the abundance of miR-182 in MGC-803 and AGS cells transfected with pcDNA, circNRIP1, si-NC or si-circNRIP1. ** P

    Article Snippet: At 48 h after the transfection, luciferase activity assay was performed in each group with a luciferase reporter assay kit (Promega).

    Techniques: Binding Assay, Luciferase, Reporter Assay, Transfection, Quantitative RT-PCR

    MXDs require SID domains to activate and repress genes (A) DNA binding of MXDs does not require SID domains. ChIP-qPCR analysis of showing that DOX-induced (24 hours) WT and ΔSID versions of MNT, MXD1, and MXD2 bind to NCL and p21 genes in U2OStx cells (n=3). (B , C) The SID domains of MXDs are required for repression of 6xEbox-luc and activation of p21-luc. (B) Luciferase reporter assay of 6xEbox-luc in HEK293 cells transfected with wild type or ΔSID MNT, MXD1, and MXD2 (n=3). (C) p21-luc expression in HEK293 cells transfected with MIZ1 and co-transfected with wild type or ΔSID versions of MXDs (n=3). * P

    Journal: bioRxiv

    Article Title: MXD/MIZ1 complexes activate transcription of MYC-repressed genes

    doi: 10.1101/842799

    Figure Lengend Snippet: MXDs require SID domains to activate and repress genes (A) DNA binding of MXDs does not require SID domains. ChIP-qPCR analysis of showing that DOX-induced (24 hours) WT and ΔSID versions of MNT, MXD1, and MXD2 bind to NCL and p21 genes in U2OStx cells (n=3). (B , C) The SID domains of MXDs are required for repression of 6xEbox-luc and activation of p21-luc. (B) Luciferase reporter assay of 6xEbox-luc in HEK293 cells transfected with wild type or ΔSID MNT, MXD1, and MXD2 (n=3). (C) p21-luc expression in HEK293 cells transfected with MIZ1 and co-transfected with wild type or ΔSID versions of MXDs (n=3). * P

    Article Snippet: Next day, luciferase expression was measured using Dual-luciferase Reporter Assay (Promega) and an EnSpire Reader (Perkin Elmer).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activation Assay, Luciferase, Reporter Assay, Transfection, Expressing

    BNIP3 is negatively regulated by miR-27a-3p. (A) The interaction between miR-27a-3p and BNIP3 was assessed by dual luciferase reporter assay, and the expression levels of miR-27a-3p, BNIP3 mRNA, and BNIP3 protein were examined by RT-qPCR and western blot analysis, respectively. ** P

    Journal: International Journal of Molecular Medicine

    Article Title: lncRNA DGCR 5/miR-27a-3p/BNIP3 promotes cell apoptosis in pancreatic cancer by regulating the p38 MAPK pathway

    doi: 10.3892/ijmm.2020.4632

    Figure Lengend Snippet: BNIP3 is negatively regulated by miR-27a-3p. (A) The interaction between miR-27a-3p and BNIP3 was assessed by dual luciferase reporter assay, and the expression levels of miR-27a-3p, BNIP3 mRNA, and BNIP3 protein were examined by RT-qPCR and western blot analysis, respectively. ** P

    Article Snippet: The luciferase activity was detected using the dual luciferase reporter assay system (Promega Corp.) according to manufacturer instructions, followed by normalizing to Renilla luciferase activity.

    Techniques: Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot

    DEDa leads to increased amount of active caspase-8 and sensitizes HeLa cells to TRAIL-induced apoptosis. ( A ) SH-SY5Y and A549 cells were separately cotransfected with pGL3-C8(−470∼+76) (1 μg) and an equal amount (0.5 μg)

    Journal: The EMBO Journal

    Article Title: Death effector domain DEDa, a self-cleaved product of caspase-8/Mch5, translocates to the nucleus by binding to ERK1/2 and upregulates procaspase-8 expression via a p53-dependent mechanism

    doi: 10.1038/sj.emboj.7601571

    Figure Lengend Snippet: DEDa leads to increased amount of active caspase-8 and sensitizes HeLa cells to TRAIL-induced apoptosis. ( A ) SH-SY5Y and A549 cells were separately cotransfected with pGL3-C8(−470∼+76) (1 μg) and an equal amount (0.5 μg)

    Article Snippet: Different caspase-8 promoter fragments were generated by PCR amplification using genomic DNA isolated from HeLa cells as template and further cloned into the pGL3-Basic luciferase reporter vector (Promega).

    Techniques:

    STRA6 is a direct target of miR-873. a Putative binding sites in 3′-UTR of STRA6 for the related miRNA binding. b The mRNA expression of STRA6 after transfecting with miR-873-mimics, miR-874-mimics and miR-149-mimics in MGC803. c The protein level of STRA6 were determined by western blot after transfection. d Wild type (WT) and Mutant type (MUT) STRA6 3′UTR sequences were cloned into pGL3 luciferase reporter vector. e and f miR-873 inhibited the relative luciferase activity in GC cells co-transfecting with miR-873-mimics and pGL3-STRA6-WT. (* p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: STRA6 exerts oncogenic role in gastric tumorigenesis by acting as a crucial target of miR-873

    doi: 10.1186/s13046-019-1450-2

    Figure Lengend Snippet: STRA6 is a direct target of miR-873. a Putative binding sites in 3′-UTR of STRA6 for the related miRNA binding. b The mRNA expression of STRA6 after transfecting with miR-873-mimics, miR-874-mimics and miR-149-mimics in MGC803. c The protein level of STRA6 were determined by western blot after transfection. d Wild type (WT) and Mutant type (MUT) STRA6 3′UTR sequences were cloned into pGL3 luciferase reporter vector. e and f miR-873 inhibited the relative luciferase activity in GC cells co-transfecting with miR-873-mimics and pGL3-STRA6-WT. (* p

    Article Snippet: Dual-luciferase reporter assay The 3′-UTR sequences of STRA6 containing wild-type or mutated miR-873 binding sites were synthesised and cloned into a pGL3 luciferase reporter vector (Promega, USA).

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

    Effect of PXR activators on NF-κB-dependent gene expression in U397-3XκB-LUC cells. (A) Left panel, RT-PCR for PXR and GAPDH in U937-3XκB-LUC cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).

    Journal: The Journal of Steroid Biochemistry and Molecular Biology

    Article Title: The PXR is a drug target for chronic inflammatory liver disease

    doi: 10.1016/j.jsbmb.2010.04.012

    Figure Lengend Snippet: Effect of PXR activators on NF-κB-dependent gene expression in U397-3XκB-LUC cells. (A) Left panel, RT-PCR for PXR and GAPDH in U937-3XκB-LUC cells, Hep-G2 cells and human skin fibroblasts (Skin FB). Each lane is amplified product from the equivalent 50 ng template total RNA per well, 35 PCR cycles). (B) Luciferase activity after 5 h treatment with the indicated compounds (DMSO, 0.5% (v/v); RIF, 20 μM; HYP, 1 μM; METYR, 250 μM; PCN 20 μM; SULF 1 mM; IKK2-In 10 μM; GT, 3 μM) normalised to LPS + DMSO vehicle activity. Data are the mean and standard deviation of 3 determinations from the same experiment, typical of at least 4 separate experiments. *Significantly different ( P > 95%) reporter gene expression versus LPS-induced DMSO control using Student's t -test (two tailed). (C) Trypan blue exclusion. Upper panels, light micrographs of typical views 5 h after treatment with 300 ng/ml LPS and the indicated compound. Lower panel, percentage trypan blue exclusion in U937-3XκB-LUC cells treated with 300 ng/ml LPS and the indicated concentration of compound for 5 h (note, grey bars are the concentrations employed to examine effects on NF-κB activity in B). Data are the mean of 3 separate incubations from the same experiment, typical of 3 separate experiments. Insol, compound became insoluble when added to culture medium. *Significantly different ( P > 95%) trypan blue exclusion versus LPS-induced vehicle control (DMSO) using Student's t -test (two tailed). Similar results were obtained with compounds without the addition of LPS (data not shown).

    Article Snippet: Luciferase expression in U937-3XκB-LUC cells stably transfected with luciferase was determined also using the Dual Luc kit.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Luciferase, Activity Assay, Standard Deviation, Two Tailed Test, Concentration Assay

    CsA is an activator of the human PXR. (A) Hep-G2 cells cultured in 24-well plates were transfected with 0.5 μg (ER6) 3 -pGL3promoter and 0.05 μg RL-TK/well and PXR activation determined [6] . Data are the mean and standard deviation of at least 3 separate transfections from the same experiment expressed as fold normalised luciferase expression versus DMSO vehicle control, typical of at least 3 separate experiments. *Significantly different ( P > 95%) luciferase activity versus DMSO using Student's t -test (two tailed). (B) Human hepatocytes were isolated and cultured as outlined in Methods section and treated with the indicated compounds after the first 24 h of culture ( T 0 ) for a further 48 h prior to isolation and analysis of CYP3A4 and β-actin levels by Western blotting. Medium and treatments were replenished every 24 h. (C) RT-PCR for the indicated transcripts in human liver, two separate human hepatocyte preparations (freshly isolated cells) and two separate LS180 cultures (amplified product from the equivalent 100 ng template total RNA per well, 35 PCR cycles). (D) Expression of CYP3A4 mRNA in LS180 cells. LS180 cells were treated with the indicated compound DMSO, 0.1% (v/v); RIF, 20 μM RIF (for 48 h) and CsA and FK506 at the indicated concentrations for 72 h. Culture media and treatments were changed daily. RNA was isolated and transcript CYP3A4 and 18S rRNA levels determined by quantitative RT-PCR. *Significantly different ( P > 95%) transcript level activity versus DMSO using Student's t -test (two tailed).

    Journal: The Journal of Steroid Biochemistry and Molecular Biology

    Article Title: The PXR is a drug target for chronic inflammatory liver disease

    doi: 10.1016/j.jsbmb.2010.04.012

    Figure Lengend Snippet: CsA is an activator of the human PXR. (A) Hep-G2 cells cultured in 24-well plates were transfected with 0.5 μg (ER6) 3 -pGL3promoter and 0.05 μg RL-TK/well and PXR activation determined [6] . Data are the mean and standard deviation of at least 3 separate transfections from the same experiment expressed as fold normalised luciferase expression versus DMSO vehicle control, typical of at least 3 separate experiments. *Significantly different ( P > 95%) luciferase activity versus DMSO using Student's t -test (two tailed). (B) Human hepatocytes were isolated and cultured as outlined in Methods section and treated with the indicated compounds after the first 24 h of culture ( T 0 ) for a further 48 h prior to isolation and analysis of CYP3A4 and β-actin levels by Western blotting. Medium and treatments were replenished every 24 h. (C) RT-PCR for the indicated transcripts in human liver, two separate human hepatocyte preparations (freshly isolated cells) and two separate LS180 cultures (amplified product from the equivalent 100 ng template total RNA per well, 35 PCR cycles). (D) Expression of CYP3A4 mRNA in LS180 cells. LS180 cells were treated with the indicated compound DMSO, 0.1% (v/v); RIF, 20 μM RIF (for 48 h) and CsA and FK506 at the indicated concentrations for 72 h. Culture media and treatments were changed daily. RNA was isolated and transcript CYP3A4 and 18S rRNA levels determined by quantitative RT-PCR. *Significantly different ( P > 95%) transcript level activity versus DMSO using Student's t -test (two tailed).

    Article Snippet: Luciferase expression in U937-3XκB-LUC cells stably transfected with luciferase was determined also using the Dual Luc kit.

    Techniques: Cell Culture, Transfection, Activation Assay, Standard Deviation, Luciferase, Expressing, Activity Assay, Two Tailed Test, Isolation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Quantitative RT-PCR

    miR-135b targets GSK3β. (A) Schematic of predicted miR-135b site in the 3′UTR of GSK3β mRNA, which broadly conserved among vertebrates. (B) GSK3β expression was much lower in U87R cells compared to U87 cells at protein level and mRNA level by qRT-PCR and Western blot. (C) GSK3β expression was much lower in U87R cells compared to U87 cells at protein level by immunofluorescence. (D) miR-135b expression interference negatively regulated GSK3β protein expression by Western blot. (E) miR-135b expression interference negatively regulated GSK3β protein expression by immunofluorescence. (F) Luciferase reporter assays were performed after transfection with indicated pMIR-Report plasmids and a renilla transfection control plasmid, co-transfected with miR-135, or relevant scramble controls. Data shown were the mean±SD of six replicates and were representative of three independent experiments. *p

    Journal: PLoS ONE

    Article Title: miR-135b Contributes to the Radioresistance by Targeting GSK3β in Human Glioblastoma Multiforme Cells

    doi: 10.1371/journal.pone.0108810

    Figure Lengend Snippet: miR-135b targets GSK3β. (A) Schematic of predicted miR-135b site in the 3′UTR of GSK3β mRNA, which broadly conserved among vertebrates. (B) GSK3β expression was much lower in U87R cells compared to U87 cells at protein level and mRNA level by qRT-PCR and Western blot. (C) GSK3β expression was much lower in U87R cells compared to U87 cells at protein level by immunofluorescence. (D) miR-135b expression interference negatively regulated GSK3β protein expression by Western blot. (E) miR-135b expression interference negatively regulated GSK3β protein expression by immunofluorescence. (F) Luciferase reporter assays were performed after transfection with indicated pMIR-Report plasmids and a renilla transfection control plasmid, co-transfected with miR-135, or relevant scramble controls. Data shown were the mean±SD of six replicates and were representative of three independent experiments. *p

    Article Snippet: Construction of luc-UTR vectors The full-length GSK3β 3′-UTR was cloned into the EcoRI and HindIII sites of the pMIR-REPORT luciferase vector (Ambion, Austin, TX, U.S.) using PCR generated fragment.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Luciferase, Transfection, Plasmid Preparation