In total, 293 T cells transfected with pSIN vector containing FLAG- ERα fragment were seeded in 96-well plate and transfected with ASB10 promoter-containing luciferase reporter psiCHECK-2 (Addgene). After transfection for 24 h, the luciferase activity was measured according to the instructions of Dual-Glo Luciferase Assay kit (Promega).
Psicheck 2
Manufactured by Addgene
PsiCHECK-2 is a dual-luciferase reporter vector system used for gene expression analysis. It contains both Renilla and Firefly luciferase reporter genes, allowing for normalization of experimental data.
Automatically generated - may contain errors
3 protocols using psicheck 2
1
Transfection and Luciferase Assay for ERα Signaling
2
Investigating TGF-β1 Regulation by miR-93
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The 3′UTR and coding sequence (CDS) of TGF-β1 were amplified from the genomic DNA of human brain tissue and subcloned into the pcDNA4.0 (Addgene; cat. no. MLCC1153; 5.1kb) or psiCHECK2 (Addgene; cat. no. P0197; 6.3kb) dual luciferase reporter plasmids. Specifically, pcDNA4.0 luciferase reporters containing TGF-β1-CDS (1,173 bp), TGF-β1-3′UTR-WT (729 bp), or TGF-β1-3′UTR-MUT (729 bp; TGF-β1 with a mutated 3′UTR sequence) were constructed. The psiCHECK2 reporters contained either Nrf2 3′UTR (486 bp) or TGF-β1 3′UTR (729 bp). The constructed vectors were transfected into 293T cells and HMO6 cells with Lipofectamine® 3000 Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.). For some experiments, the luciferase reporter plasmids were co-transfected with an miR-93 mimic (or the control; mimic NC), miR-93 inhibitor (or the control; inhibitor NC), siTGF-β1 (5′-AAGGGCTACCATGCCAACTTC-3′) (33 (link)), or the scramble control siTGF-β1-NC, purchased from RiboBio (Guangzhou RiboBio Co., Ltd.). Luciferase activity was analyzed as previously described (34 (link)). Each assay was performed in triplicate.
3
Molecular Cloning and Plasmid Construction
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The pBABE-miR-31 plasmid (Plasmid#26088), pWPXL-SOX4 (plasmid#36984), pCMVHA-hEZH2 (plasmid#24230), psicheck2 SOX4 full-length 3′UTR (Plasmid#26989) were purchased from Addgene (Cambridge, MA).
A 71 bp WT fragment of the SOX4 3′UTR (SOX4 WT OLIGO) was created by overlapping extension PCR and cloned between the XhoI and NotI site of the psicheck2 plasmid. Similarly, the mutant construct of SOX4 3′UTR (SOX4 Mutant OLIGO) which carried a substitution of four nucleotides within the core seed sequence of miR-31, was carried out using overlapping extension PCR and cloned between the XhoI and NotI site of the psicheck2 plasmid.
The two set of plasmids containing shRNA specific to SOX4 and EZH2 were purchased from OriGene (Rockville, MD).
Primers used for SOX4, forward: 5′-AGCGACAAGATCCCTTTCATTC-3′, reverse: 5′-CGTTGCCGGACTTCACCTT-3′; EZH2, forward: 5′-GTACACGGGGATAGAGAATGTGG-3′, reverse: 5′GGTGGGCGGCTTTCTTTATCA-3′, for EZH1, forward: 5′-ATGCGACTTCGACAACTTAAACG-3′, reverse: 5′-GGCTTCATTGACTGAACAGGTT-3′, HDAC3, forward: 5′-CCTGGCATTGACCCATAGCC-3′, reverse: 5′-CTCTTGGTGAAGCCTTGCATA-3′, GAPDH, forward: 5′-GCGACACCCACTCCTCCAC-3′, reverse: 5′-TCCACCACCCTGTTGCTGTAG-3′.
A 71 bp WT fragment of the SOX4 3′UTR (SOX4 WT OLIGO) was created by overlapping extension PCR and cloned between the XhoI and NotI site of the psicheck2 plasmid. Similarly, the mutant construct of SOX4 3′UTR (SOX4 Mutant OLIGO) which carried a substitution of four nucleotides within the core seed sequence of miR-31, was carried out using overlapping extension PCR and cloned between the XhoI and NotI site of the psicheck2 plasmid.
The two set of plasmids containing shRNA specific to SOX4 and EZH2 were purchased from OriGene (Rockville, MD).
Primers used for SOX4, forward: 5′-AGCGACAAGATCCCTTTCATTC-3′, reverse: 5′-CGTTGCCGGACTTCACCTT-3′; EZH2, forward: 5′-GTACACGGGGATAGAGAATGTGG-3′, reverse: 5′GGTGGGCGGCTTTCTTTATCA-3′, for EZH1, forward: 5′-ATGCGACTTCGACAACTTAAACG-3′, reverse: 5′-GGCTTCATTGACTGAACAGGTT-3′, HDAC3, forward: 5′-CCTGGCATTGACCCATAGCC-3′, reverse: 5′-CTCTTGGTGAAGCCTTGCATA-3′, GAPDH, forward: 5′-GCGACACCCACTCCTCCAC-3′, reverse: 5′-TCCACCACCCTGTTGCTGTAG-3′.
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