786-O and ACHN cells were co-transfected with a Renilla luciferase plasmid containing the promoter region of ZO-1 (Active Motif) and pCMVHA hEZH2 (Addgene) or pCMV6-Entry (Origene). Promoter activity was determined using a LightSwitch Luciferase Assay Kit (Active Motif) according to the manufacturer’s instructions. pCMVHA hEZH2 was a gift from Kristian Helin (Addgene plasmid # 24230; http://n2t.net/addgene:24230 ; RRID:Addgene_24230) [29 (link)].
Pcmvha hezh2
Manufactured by Addgene
PCMVHA hEZH2 is a plasmid that expresses the human Enhancer of Zeste Homolog 2 (EZH2) protein under the control of the CMV promoter. EZH2 is a core component of the Polycomb Repressive Complex 2 (PRC2) and plays a role in epigenetic regulation through histone methylation.
Automatically generated - may contain errors
Lab products found in correlation
Lightswitch luciferase assay kit, by Active Motif (1 mentions)
Pcmv6 entry, by OriGene (1 mentions)
Lmax luminometer, by Molecular Devices (1 mentions)
Dual luciferase assay system protocol, by Promega (1 mentions)
Prl tk renilla luciferase construct, by Promega (1 mentions)
Pgl3 basic vector, by Promega (1 mentions)
Saci and xhoi restriction enzymes, by New England Biolabs (1 mentions)
Pcdna3 mrfp, by Addgene (1 mentions)
Pcmv vsv g, by Addgene (1 mentions)
Pcmv dr8.2 dvpr, by Addgene (1 mentions)
Plko 1 shgfp, by Addgene (1 mentions)
Prl sv40, by Promega (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Psicheck 2, by Addgene (1 mentions)
Gapdh, by OriGene (1 mentions)
6 protocols using pcmvha hezh2
1
ZO-1 Promoter Activity Assay
2
Plasmid Transfection and Protein Analysis
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The A549 cell line was transfected with 10 μg of EZH2 (pCMVHA hEZH2, RRID: Addgene_24230) and PHD3 (plp6.3-Egln3, RRID: Addgene_79115) plasmid using Lipofectamine 3000 (Thermo F. Scientific, #L3000015). Each plasmid was transfected either individually or in combination. After three days, cells were harvested, lysed with RIPA buffer, and analyzed by SDS-PAGE and Western blotting.
3
RelB Promoter Luciferase Assay
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The RelB promoter containing 898 base pairs of sequence directly upstream of the start site was cloned into the PGL3-Basic vector (Promega) using SacI and XhoI restriction enzymes (New England Biolabs). 293 cells were transfected with 500 ng of the RelB reporter construct, 50 ng pRL-TK Renilla luciferase construct (Promega) and 500 ng of either pFLAG-CMV-p65 or pCMVHA-hEZH2 (Addgene, #24230) using polyethylenimine linear (Sigma-Aldrich). The cells were harvested 24 hours later in passive lysis buffer and analyzed according to Dual-Luciferase Assay System protocol (Promega). Activity was measured using an Lmax Luminometer (Molecular Devices) and relative light units were normalized to pRL-TK Renilla luciferase light units.
4
Plasmid Cloning and Validation
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pBJ-FLAG-hPar4 (cat #53231), pCMVHA hEZH2 (cat #24230), pcDNA3-mRFP (cat #13032), pCMV-VSV-G (cat #8454) and pCMV-dR8.2 dvpr (cat #8455) plasmids were purchased from Addgene. Human PAR2 (hPar2/ f2rl1) plasmid was kindly provided by Dr. Morley D. Hollenberg (Faculty of Medicine, University of Calgary, Calgary, AB, Canada). flg-β-catenin and flg-Axin were kindly provided by Dr. Ben-Neriah (Hebrew University, Jerusalem, Israel). All of the mentioned plasmids were sequenced to confirm the absence of undesirable mutations. Details of the plasmids are available on request.
5
Lentiviral Knockdown and Overexpression
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Cisplatin and chemical reagents, including Tris, NaCl, SDS and DMSO, for molecular biology and buffer preparation were purchased from Sigma-Aldrich (St. Louis, MO). Lentivirus plasmids containing pLKO.1-shEZH2 (#1, TRCN0000040073; #2, TRCN0000040074; #3, TRCN0000040075; #4, TRCN0000040076; #5, TRCN0000040077), pLKO.1-shSUZ12 (#1, TRCN0000038724; #2, TRCN0000038725; #3, TRCN0000038726; #4, TRCN0000038727; #5, TRCN0000038728), and pLKO.1-shEED (#1, TRCN0000021204; #2, TRCN0000021205; #3, TRCN0000021206; #4, TRCN0000021207; #5, TRCN0000021208) were purchased from Thermo Scientific. The pCMV-HA vector (cat#631604) was purchased from CloneTech. The EZH2 expression construct pCMV-HA-hEZH2 (Addgene plasmid #24230), the luciferase reporter Puma-Luc (Addgene plasmid #16591), the pBV-Luc construct (Addgene plasmid #16539), pLKO.1-shGFP (Addgene plasmid #30323), the lentiviral packaging plasmid psPAX2 (Addgene plasmid #12260) and the envelope plasmid pMD2.G (Addgene plasmid #12259) were available on Addgene (Cambridge, MA). The Renilla luciferase reporter construct pRL-SV40 (Promega) was used as previously described [59 (link)].
6
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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