TP53 promoter activity was assessed using a dual-luciferase reporter assay, following a previously described method [18 (link)]. Briefly, to investigate the effect on TP53 promoter activity, cells were cotransfected with reporter constructs including pGL3.0-basic (Promega, Madison, WI, USA) and TP53-luciferase, along with the pNL1.1.TK vector. After 24 h of transfection, luciferase activity was measured using the Nano-Glo® Dual Luciferase® Reporter Assay System (Promega), following the manufacturer’s protocols. The relative firefly luciferase activity was normalized to NanoLuc™ luciferase activity to account for variations in transfection efficiency.
Pgl3.0 basic
Manufactured by Promega
Sourced in United States
PGL3.0-Basic is a plasmid vector designed for gene expression studies. It contains a multiple cloning site for inserting genes of interest and a firefly luciferase reporter gene for detecting and quantifying gene expression. The plasmid also includes an origin of replication and an antibiotic resistance marker for selection in bacterial hosts.
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Lab products found in correlation
Dual luciferase reporter assay system, by Promega (7 mentions)
Enspire 2300 multilabel reader, by PerkinElmer (6 mentions)
Nano glo dual luciferase reporter assay system, by Promega (2 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Pnl1.1 tk vector, by Promega (1 mentions)
Lipofectamine ltx reagent, by Thermo Fisher Scientific (1 mentions)
Site directed mutagenesis kit, by Takara Bio (1 mentions)
Dual luciferase assay system, by Promega (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
E coli dh5α competent cells, by Tiangen Biotech (1 mentions)
Gel extraction kit, by Tiangen Biotech (1 mentions)
Tamibarotene, by Merck Group (1 mentions)
Prl sv40, by Promega (1 mentions)
12 protocols using pgl3.0 basic
1
TP53 Promoter Activity Assay
2
Luciferase assay for CDX2 promoter
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Fragments of the CDX2 were amplified PCR using primers (Supplementary Table S1 ) and cloned into the luciferase reporter vector pGL3.0-Basic (Catalog: E1751, Promega, Madison, WI, USA) to generate CDX2 promoter reporter constructs. Plasmids containing firefly luciferase reporters and JARID1B plasmids were cotransfected into cells. For the TOP/FOP-Flash reporter assay [24 (link)], the TOP/FOP-Flash reporter and JARID1B plasmids were co-transfected into cells. After transfection for 48 h, the cells were harvested for analysis with the Dual-Luciferase Reporter Assay System (Catalog: E1910, Promega, Madison, WI, USA). Luciferase activity was measured using the PerkinElmer EnSpire Multilabel Reader 2300 (PerkinElmer Inc., Waltham, MA, USA). The luciferase intensity was normalized to the Renilla luciferase activity to normalize for transfection efficiency.
3
Dual Luciferase Assay for YAP/TEAD Transcriptional Activity
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Cells were cotransfected with 100 ng of each reporter construct and 0.25 ng of the pNL1.1.TK vector (Promega, Madison, WI, USA) per well in white-bottom 96-well plates (SPL Life Science, Pocheon, Korea) using 0.5 µl of FuGENE® HD Transfection Reagent (Promega). To determine the effect of YAP/TEAD promoter activity, cells in each well were cotransfected with reporter constructs such as pGL3.0-basic (Promega) and 8xGITTC-luciferase (Addgene, plasmid #34615, Cambridge, MA, USA) and the pNL1.1.TK vector. Forty-eight hours post transfection, luciferase activity was measured with a Nano-Glo® Dual Luciferase® Reporter Assay System (Promega) according to the manufacturer’s recommendations. Relative firefly luciferase activity was normalized to NanoLuc™ luciferase activity to adjust for variations in the transfection efficiency.
4
Luciferase Reporter Assay for Wnt Pathway
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Fragments of the Axin2 (from position –2508 to +153 bp) and the GSK-3β 5′-flanking sequence (from position –222 to +180 bp) were amplified PCR using primers (Table 1 ) using primers (Table 1 ) and cloned into the luciferase reporter vector pGL3.0-Basic (Promega, Madison, WI, USA) to generate Axin2 and GSK-3β promoter reporter constructs. The AXIN2 enhancer region (position −15,083 to −16,104 bp) was cloned into Axin2 promoter reporter constructs. Plasmids containing firefly luciferase reporters and pTK-RL plasmids were co-transfected into cells. For the TOP/FOP-Flash reporter assay, the TOP/FOP-Flash reporter and pTK-RL plasmids were co-transfected into cells. After transfection for 48 h, the cells were harvested for analysis with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Luciferase activity was measured using the PerkinElmer EnSpire Multilabel Reader 2300 (PerkinElmer Inc., Waltham, MA, USA). The luciferase intensity was normalized to the Renilla luciferase activity to normalize for transfection efficiency.
5
Transfection of FLAG-tagged ET-1 Plasmids
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Human expression plasmids for FLAG-tagged ET-1 and Lys198Asn polymorphism were cloned between MluI and BglII sites in the firefly luciferase reporter vector pGL3.0 Basic (Promega) using standard molecular biology techniques. Briefly, 1×105 HEK-293T cells per well were added to 0.5 mL of RPMI 1640 supplemented with 100 U/mL penicillin, 100 ug/mL streptomycin, 2 mM glutamine, 5 uM 2-ME (Sigma-Aldrich), and 10% fetal bovine serum. Cell density was 50–80% confluent on the day of transfection. We added 0.75–1.75 μL of Lipofectamine LTX Reagent (ThermoFisher Scientific) to the DNA sample (0.5 μg of DNA diluted in 100 μL of Reduced Serum Media without serum), mixed gently and incubated for 30 minutes at room temperature. Then 100 μL of the DNA-Lipofectamine complex was added to each well containing cells and mixed gently. The cells were incubated at 37oC in a humidified CO2 incubator for 18–24 hours post-transfection before assaying for transgene expression. Cells were incubated in the presence of 10 ng/mL and 20 ng/mL of each of TNF-α, IL-6, or LPS for an additional 24 hours. Cell lysates were prepared for western blot analysis.
6
Construction of SHP Promoter Reporters
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Fragments of the SHP 5′-flanking sequence were amplified by PCR using special primers (Table S1 ) and cloned into the luciferase reporter vector pGL3.0-Basic (Promega, Madison, WI, USA) to generate SHP promoter reporter constructs (wild-type SHP promoter, SHP-WT promoter). Mutagenesis of FXR binding site in SHP promoter (SHP-mFXR promoter) was performed using a site-directed mutagenesis kit (Takara Biotechnology Co., Ltd.). The detailed protocol was performed as described previously32 (link). Each experiment was performed in triplicate.
7
Regulation of CYP3A11 Gene Expression
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The promoter region of CYP3A11 genes was amplified by PCR and subcloned into pGL3.0-basic (Promega). These constructs were introduced into hepatocytes along with pSV-TK(Promega), which was used to normalize the transfection efficiency. Then cells were treated with PJ34 or APAP as indicated and luciferase activities were measured 24 h later using the Dual-Luciferase Assay System (Promega).
8
Constructing pEGFP-N1 Vector in DH5α Cells
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The pEGFP-N1 vector was constructed in our laboratory. E. coli DH5α competent cells, gel extraction kit, and miniprep kits were purchased from the Tiangen Company (Beijing, China). DL5000 DNA Marker Prime STARMax DNA Polymerase, T4 DNA ligase, and restriction endonuclease were purchased from Takara (Dalian, China). The expression vector pGL3.0-Basic, pRL-SV40, and the Dual-Luciferase Reporter Assay System were purchased from Promega Corporation. LipofectamineTM2000 was purchased from Invitrogen Corporation. ATRA, Tamibarotene (Tamibarotene, Am80), and TSA were purchased from SIGMA Company. The chicken embryo fibroblast cell line, DF-1, and Mouse spermatogonial cell line, GC-1,were purchased from ATCC. Primer synthesis and sequencing were conducted by the Invitrogen Company of Shanghai (China).
9
CDX2 Promoter Regulation Assay
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Fragments of the CDX2 were ampli ed PCR using primers (Supplementary Table S2) and cloned into the luciferase reporter vector pGL3.0-Basic (Catalog: E1751, Promega, Madison, WI, USA) to generate CDX2 promoter reporter constructs. Plasmids containing re y luciferase reporters and JARID1B plasmids were cotransfected into cells. For the TOP/FOP-Flash reporter assay [25] , the TOP/FOP-Flash reporter and JARID1B plasmids were co-transfected into cells. After transfection for 48 h, the cells were harvested for analysis with the Dual-Luciferase Reporter Assay System (Catalog: E1910, Promega, Madison, WI, USA). Luciferase activity was measured using the PerkinElmer EnSpire Multilabel Reader 2300 (PerkinElmer Inc., Waltham, MA, USA). The luciferase intensity was normalized to the Renilla luciferase activity to normalize for transfection e ciency.
10
CDX2 Promoter Regulation Assay
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Fragments of the CDX2 were ampli ed PCR using primers (Supplementary Table S2) and cloned into the luciferase reporter vector pGL3.0-Basic (Catalog: E1751, Promega, Madison, WI, USA) to generate CDX2 promoter reporter constructs. Plasmids containing re y luciferase reporters and JARID1B plasmids were cotransfected into cells. For the TOP/FOP-Flash reporter assay [25] , the TOP/FOP-Flash reporter and JARID1B plasmids were co-transfected into cells. After transfection for 48 h, the cells were harvested for analysis with the Dual-Luciferase Reporter Assay System (Catalog: E1910, Promega, Madison, WI, USA). Luciferase activity was measured using the PerkinElmer EnSpire Multilabel Reader 2300 (PerkinElmer Inc., Waltham, MA, USA). The luciferase intensity was normalized to the Renilla luciferase activity to normalize for transfection e ciency.
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