Transient transfection and reporter gene luciferase assays were carried out as previously described. 43 (link),56 (link) Briefly, 10T1/2 cells or HEK293T cells were maintained in DMEM/10% FBS with 1% penicillin and streptomycin. Then the cells were seeded in 12-well plates one day before transient transfection. Sub-confluent cells were co-transfected using Lipofectamine 3000 (ThermoFisher) with the luciferase reporter construct (in pGL3B vector, Promega) harboring SM a-actin gene promoter (nucleotide positions −2555 to +2813 containing CArG regions as reported before 57 (link)), CMV-Renilla luciferase reporter as an internal control (Promega), along with or without SMYD2 and/or myocardin expression plasmids. Post-transfection 24-48 h, cell lysates were collected, and the luciferase activity was determined using the Dual-Luciferase Reporter Assay System kit (Promega) by a plate reader according to the manufacturer’s instruction. The firefly luciferase activity values in the lysates were normalized to the TK Renilla luciferase reporter activity as the internal control for transfection efficiency. The data were presented as the fold changes relative to empty vector or myocardin alone (set to 1). A minimum of 3 independent transfections was performed and all assays were replicated at least three times.
Pgl3b vector
Manufactured by Promega
Sourced in United States
The PGL3B vector is a plasmid designed for use in luciferase reporter gene assays. It contains the firefly luciferase gene under the control of a minimal promoter element, which can be used to measure transcriptional activity in a variety of cell lines and experimental systems.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (4 mentions)
Cmv renilla luciferase reporter, by Promega (1 mentions)
Dual luciferase reporter assay system kit, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
Quikchange lightning site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Iproof high fidelity dna polymerase, by Bio-Rad (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
Xl10 gold ultracompetent cells, by Agilent Technologies (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
5 protocols using pgl3b vector
1
Promoter Activity Regulation Assay
2
KLF5 Binding Site Analysis of CFTR Enhancer
3
Generating Hybrid Promoter Constructs for Gene Expression Analysis
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The pDRAprox vector has been previously described (Krawczyk et al., 2007 (link)). This vector contains an HLA-DRA promoter fragment (from −151 to +10) inserted upstream of the firefly luciferase reporter gene in the pGL3b vector (Promega). The vector containing the BTN2A2 promoter was generated by inserting a PCR-amplified fragment encompassing the SXY module and transcription start site of BTN2A2 between the MluI and BglII sites of pGL3b. Vectors containing hybrid promoters were generated by replacing the HLA-DRA SXY module with BTN SXY fragments using the MluI–BglII sites in pDRAprox. Mutations were introduced as previously described (Krawczyk et al., 2007 (link)). Primers used for cloning are available upon request. Raji, RJ2.2.5, and SJO cells were cotransfected with the firefly luciferase reporter vectors and a control renilla luciferase vector. Transfections were performed by electroporation (950 µF and 0.21 V). Dual luciferase assays were performed as previously described (Krawczyk et al., 2007 (link)).
4
Cloning and Mutagenesis of SCD5 Regulatory Region
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Different sized fragments of SCD5 upstream regulatory region were amplified using iProof™ High-Fidelity DNA Polymerase (Bio-Rad, Hercules, CA, USA) from human genomic DNA template using primers that contain Kpn I and Hind III restriction endonuclease recognition sites. After purification and restriction endonuclease digestion (Thermo Fisher Scientific, Waltham, MA, USA), the amplicons were ligated (T4 Ligase, Thermo Fisher Scientific, Waltham, MA, USA) into pGL3B vector (Promega, Madison, WI, USA) that was upstream of the luciferase reporter gene. The studied natural variants were generated using Q5® Site-Directed Mutagenesis Kit (New England BioLabs, Ipswich, MA, USA) following the manufacturer’s instruction. Mutagenic primers were designed using the online NEB primer design software, NEBaseChanger™. After digesting the original nonmutated and methylated plasmid by KLD reaction, an aliquot of constructs was transformed into XL10-Gold® Ultracompetent Cells (Agilent, Santa Clara, CA, USA), which were then screened for positive colonies by single cell PCR. The cloning and mutagenic primers are listed in Table S1 . All of the constructs were verified by Sanger sequencing them.
5
Regulation of ELAVL3 Promoter and ALK Expression in Ovarian Cancer
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The human ELAVL3 promoter, encompassing e2321 to 77 bp (where 1 represents the transcription start site), was amplified by PCR using specific primers (forward, 5 0 -CTTGATCCCTTCCTAAGCGAGGCAG-3 0 ; and reverse, 5 0 -GTCCCGTGTGTTCAAGTCCTCTCC-3 0 ), and was sub cloned into the pGL-3B vector (Promega, Madison, WI). The pGL3B-(e2056/30) ALK, pcDNA3.1efull-length ALK, pcDNA3.1-SRY-box transcription factor (Sox)2, pcDNA3.1-Sox3, pcDNA3.1-Sox4, pcDNA-Sox5, pcDNA3.1-Sox6, pcDNA3.1-Sox7, pcDNA3.1-Sox9, pcDNA3.1-Sox11, pcDNA3.1-Sox17, pCMVep53 wild type, p53 mutant type (R248Q; p53mt), and pSIREN-RetroQ-short hairpin (sh) ALK were used as described previously. 22e25 Three HGSC cell lines, OVSAHO, OVKATE, and OVCAR-3, and five ovarian clear cell carcinoma (CCC) cell lines, OVISE, ES-2, OVTOKO, KOC7C, and TOV-21 G, were used as described previously. 26e28 The full-length ALK expression plasmid or empty vector was transfected into OVCAR-3 cells (with relatively low endogenous ALK expression), and clones stably overexpressing OVCAR3 cells transfedted of ALK expression gene (CA-ALK) were established. ALK-knockdown lines were also generated using OVSAHO cells [which have relatively high ALK expression; OVSAHO cells transfected of ALK-shRNA vector (SA-shALK)] and shRNA targeting the ALK gene, as described previously. 22
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