For SP, cells were re-suspended at 1×10^6 cells/ml then incubated at 37°C for 30' with or without Fumitremorgin C (FTC) (Sigma). Cells were then incubated for a further 90' with 5 μg/ml Hoechst 33342 with periodic shaking. Hoechst 33342 was excited at 407 nm using a trigon violet laser, and dual wavelength detection was performed using 450/40 (Hoechst 33342-Blue) and 695/40 (Hoechst 33342-Red) filters. For luciferase reporter assay, cells were co-transfected with HRE-luc ((Emerling et al., 2008 (link)); Addgene #26731) and pCMV-renilla (Promega), then analyzed for luciferase using the Dual-Luciferase Reporter Assay System (Promega) on a Veritas luminometer (Turner Biosystems). For colony formation assay, cells were plated at clonal density in 6-well plates, then irradiated. Colonies were fixed and manually counted at day 10 post-irradiation.
Pcmv renilla
Manufactured by Promega
Sourced in United States
The PCMV-Renilla is a plasmid vector that contains the Renilla luciferase gene under the control of the cytomegalovirus (CMV) promoter. This plasmid can be used for the expression and detection of Renilla luciferase in various cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (7 mentions)
Synergy 2 plate reader, by Agilent Technologies (2 mentions)
X tremegene 9, by Roche (2 mentions)
Psmpuw m3 pparγ, by Promega (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle, by Thermo Fisher Scientific (2 mentions)
Lipod293, by SignaGen (2 mentions)
Veritas luminometer, by Promega (1 mentions)
Fumitremorgin c ftc, by Merck Group (1 mentions)
Hre luc, by Addgene (1 mentions)
Hiperfect, by Qiagen (1 mentions)
Pmir report vector, by Thermo Fisher Scientific (1 mentions)
Pcdna6.2 gw emgfp mir, by Thermo Fisher Scientific (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Plenti6 v5 dest, by Thermo Fisher Scientific (1 mentions)
Pdonr222, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
9 protocols using pcmv renilla
1
Evaluating Cellular Resistance Mechanisms
2
Hypoxia-Responsive Luciferase Assay
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DLK-S expression plasmid was kindly provided by prof. Anne Ferguson-Smith [11] (link), cloned into RCAS vector by classic restriction enzyme technique and transfected into DF-1 cells. For luciferase reporter assay, cells were co-transfected with hypoxia-responsive element (HRE)-luc (Addgene) [26] (link) or 8xCSL-luc (gift from Håkan Axelson) and pCMV-renilla (Promega) and analyzed using the Dual-Luciferase Reporter Assay System (Promega) on a Synergy 2 platereader (BioTek). Xtreme gene 9 (Roche) reagent was used according to manufacturer's recommendations for transient transfections.
3
Notch Signaling Pathway Activation Assay
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DLK plasmids were a kind gift from Ferguson et al. [11 (link)]. Plasmids were transfected using Xtreme gene 9 (Roche) and siRNAs using HiPerFect (QIAGEN). Non-targeting (D-001810–01–20) HIF1A (LQ-004018–00–0002) and HIF2A (LQ-004814–00–0002) siRNAs were obtained from GE Dharmacon. For the generation of U3084S stably expressing DLKs, U3084MG cells were transfected, selected using 750 µg/ml G418 and expanded. Stable pools were used without cloning.
For luciferase reporter assays, cells were co-transfected with 8xCSL-luc (gift from Håkan Axelson) and pCMV-renilla (Promega), together with indicated constructs, then analyzed using the Dual-Luciferase Reporter Assay System (Promega) on a Synergy 2 platereader (BioTek, Winooski, USA). The human Notch1-ICD plasmid was a gift from Håkan Axelson.
For Notch luciferase assay, three independent experiments were performed, read in duplicate.
For luciferase reporter assays, cells were co-transfected with 8xCSL-luc (gift from Håkan Axelson) and pCMV-renilla (Promega), together with indicated constructs, then analyzed using the Dual-Luciferase Reporter Assay System (Promega) on a Synergy 2 platereader (BioTek, Winooski, USA). The human Notch1-ICD plasmid was a gift from Håkan Axelson.
For Notch luciferase assay, three independent experiments were performed, read in duplicate.
4
APOBEC3A/B 3'UTR Regulation by miR-409
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A3A 3′UTR and A3B 3′UTR were PCR amplified from genomic DNA of OC3 oral cancer cell line, using the same forward primer 5′-ACTAGTAGGATGGGCCTCAGT CTCTAAG-3′; reverse primer 5′-AAGCTTAGTGTTTGTGGAAACTCTTGCAATT C-3′ is for A3A 3′UTR, and 5′-AAGCTTAGTGTTTGTGGAAACAATTATGGAAG-3′ is for A3B 3′UTR. The amplified products were cloned into the pMIR-Report-Vector (Ambion). Precursor of miR-409 was amplified from OC3 cell genomic DNA, and cloned into pcDNA6.2-GW/EmGFP-miR (Invitrogen). 293T cells (2 × 105) were subjected to transient calcium-phosphate-mediated transfection with the following: 10 ng of pMIR-Lusiferase-A3B 3′UTR; 1 μg of the vector control or expression vectors encoding miR-409 and 10 ng of pCMV-Renilla (Promega). Luciferase and renilla activities were measured using the Dual-Luciferase Reporter Assay System (Promega). The luciferase values were normalized to those of renilla, and the results are presented as the luciferase/renilla ratio.
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A3A 3′UTR and A3B 3′UTR were PCR amplified from genomic DNA of OC3 oral cancer cell line, using the same forward primer 5′-ACTAGTAGGATGGGCCTCAGT CTCTAAG-3′; reverse primer 5′-AAGCTTAGTGTTTGTGGAAACTCTTGCAATT C-3′ is for A3A 3′UTR, and 5′-AAGCTTAGTGTTTGTGGAAACAATTATGGAAG-3′ is for A3B 3′UTR. The amplified products were cloned into the pMIR-Report-Vector (Ambion). Precursor of miR-409 was amplified from OC3 cell genomic DNA, and cloned into pcDNA6.2-GW/EmGFP-miR (Invitrogen). 293T cells (2 × 105) were subjected to transient calcium-phosphate-mediated transfection with the following: 10 ng of pMIR-Lusiferase-A3B 3′UTR; 1 μg of the vector control or expression vectors encoding miR-409 and 10 ng of pCMV-Renilla (Promega). Luciferase and renilla activities were measured using the Dual-Luciferase Reporter Assay System (Promega). The luciferase values were normalized to those of renilla, and the results are presented as the luciferase/renilla ratio.
5
Measuring MYC-Driven Luciferase Activity
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Cells were transfected with 0.4 μg of MYC luciferase reporter plasmid (plasmid 16601) from Addgene and 0.1 μg pCMV-Renilla (Promega, Madison, WI) per well using the TransIT-2020 transfection reagent. Activation of the luciferase constructs was measured at 48 h post-transfection using the Dual Luciferase Assay Kit (Promega). Luciferase values were normalized to Renilla luminescence. Three independent experiments were performed in quadruplicate. Data are presented as the mean ± SE for all experiments.
6
Generation of Expression Vectors for KSHV Proteins
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The expression constructs for LANA (pcDNA3.1/LANA) and KSHV cluster miRNAs (pcDNA3.1/cluster) were described in previous reports [24 ,33 (link)]. For construction of vFLIP and vCyclin expression vectors, Gateway Cloning method was used. After ORFs of vFLIP and vCyclin were amplified by PCR and cloned into entry vector (pDONR222, Invitrogen) using BP recombination, ORFs were cloned into pLenti6/V5-DEST (Invitrogen) using LR recombination following the manufacturer’s procedure to create pLenti6/vFLIP and pLenti6/vCyclin. Reporter vector containing the promoter of miR-17-92 cluster upstream of luciferase vector was kindly provided from Dr. De Guire [19 (link)]. pGL3-SBE4 (Promega) contains four SMAD binding elements upstream of luciferase gene, activated by TGF-β ligand. pCMV-Renilla (Promega), expressing renilla luciferase, was used for normalization of firefly luciferase activity.
7
PPARγ-Mediated Luciferase Reporting in HEK293
8
PPARγ-Mediated Luciferase Reporting in HEK293
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The human HEK293 cells were cultured in 6-well plates in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Invitrogen), 100 μg/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). Luciferase reporter assays were performed by transfecting 1 μg of PPRE3x-tk-Luc reporter construct, 1 μg of pSMPUW-M3-PPARγ, pSMPUW-PPARγ or pSMPUW-GFP control, and 2 ng of pCMV-Renilla (Promega) into HEK293 cells using LipoD293 (SignaGen Laboratories, Rockville, MD). The cells were cultured in the presence or absence of rosiglitazone (1 μM) for 48 hr after transfection and lysed for luciferase and renilla (for correction) activity assay by using Dual-Luciferase Reporter Assay System (Promega, Madison, WI).
9
Transcriptional Regulation by Glucocorticoid Receptor
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COS-1 cells were seeded into 24-well plates (3 Â 10 4 cells/well). Transfection was performed 24 h later using the TransIT ® eCOS Transfection Kit (Mirus Bio) according to the manufacturer's instructions. MMTV:luciferase reporter construct (200 ng) was transfected, with a range of PCS þ zGRa plasmid concentrations (1e300 ng) and/or 100 ng pCS2þzGRb expression vector (Schaaf et al., 2008) , together with 2 ng pCMV:renilla (Promega). In a second set of experiments, cells were transfected with 50 ng of a kB:luciferase reporter construct (Stratagene) and 50 ng of a human p65 expression vector (pCMV4-p65 (Ruben et al., 1991) ), together with a range of pCS2þzGRa concentrations (0e1000 ng) in the presence and absence of 100 ng pCS2þzGRb. The total amount of transfected DNA was always kept equal among groups by transfecting empty pCS2þ vector. Twenty-four hours after transfection, cells were treated with 100 nM dexamethasone (Sigma) and 24 h later, they were assayed for luciferase activity using the Dual-Luciferase ® Reporter Assay System (Promega). Bioluminescence was detected using a Wallac 1450 MicroBeta Luminometer. For each sample, the luciferase activity was normalized to the renilla activity. Per sample, measurements were performed in duplicate, and data shown are averages ± s. e.m. of three experiments.
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