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pgl3 basic 180 luciferase reporter plasmid  (Addgene inc)


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    Addgene inc pgl3 basic 180 luciferase reporter plasmid
    Pgl3 Basic 180 Luciferase Reporter Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 95/100, based on 195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pgl3+basic/pGL3+(-BRE)+Luciferase+(Plasmid+%2345127)/10__1158_slash_0008___5472__can___25___2893-63-26-31
    Average 95 stars, based on 195 article reviews
    pgl3 basic 180 luciferase reporter plasmid - by Bioz Stars, 2026-09
    95/100 stars

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    Related Articles

    Construct:

    Article Title: β-catenin: A crucial transcriptional activator of KSHV latency genes and small molecule target in primary effusion lymphoma.
    Article Snippet: Latency-associated nuclear antigen (LANA)-induced β-catenin accumulation is one of the prime events in the Kaposi sarcoma-associated herpesvirus (KSHV)-associated primary effusion lymphoma (PEL).. LANA interacts with GSK3β, which in turn increases β-catenin pool in this lymphoma.. The intrinsic molecular mechanism and the effects of β-catenin accumulation in this lymphoma is still unclear. β-catenin acts as a major transcription factor that regulates a variety of proliferative signaling pathways and promotes oncogenesis.

    Article Title: FOSL1 Orchestrates Epigenetic Reprogramming of Anaplastic Thyroid Cancer and Suppresses Natural Killer Cell-Mediated Antitumor Immunity
    Article Snippet: 2 Anaplastic thyroid cancer (ATC) is the most aggressive type of thyroid cancer with 37 survival time of only 7-10 months.. Previous work revealed reduced proportions and 38 cytotoxicity of natural killer (NK) cells in ATC tumor microenvironment (TME).. Here, we 39 investigated the role of super-enhancers (SEs), clusters of adjacent enhancers and drive high 40 expression of genes, in reshaping the TME in ATC.

    Control:

    Article Title: β-catenin: A crucial transcriptional activator of KSHV latency genes and small molecule target in primary effusion lymphoma.
    Article Snippet: Latency-associated nuclear antigen (LANA)-induced β-catenin accumulation is one of the prime events in the Kaposi sarcoma-associated herpesvirus (KSHV)-associated primary effusion lymphoma (PEL).. LANA interacts with GSK3β, which in turn increases β-catenin pool in this lymphoma.. The intrinsic molecular mechanism and the effects of β-catenin accumulation in this lymphoma is still unclear. β-catenin acts as a major transcription factor that regulates a variety of proliferative signaling pathways and promotes oncogenesis.

    Clone Assay:

    Article Title: Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management
    Article Snippet: .. Briefly, signal peptide (SP)-HRP (secreted HRP (sHRP)) was cloned from pLNCX-Optimized mHRP-myc-mB7 vector, as described in our previous work [ ], by PCR with 5′HindIII and 3′ClaI flanking SP-HRP-myc and the transmembrane mB7 sequence was removed to obtain pLNCX-Optimized sHRP-myc. pTEAD1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc, removing mB7 and subcloning into 8xGTIIC-luciferase (Addgene plasmid #34,615). pAP- 1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc and subcloned into 3 × AP- 1 in pGL3-basic (pAP- 1-Luciferase, Addgene plasmid #40,342). pNF-κB-sHRP-myc was generated by PCR with 5′HindIII and 3′EcoRI flanking sHRP-myc from pLNCX-Optimized mHRP-myc-mB7 and subcloned into pGL4.32[luc2P/NF-κB-RE/Hygro] (E8491, Promega, Madison, US). cHRP was generated by PCR with 5′HindIII and 3′SalI to remove SP and subcloned into pLNCX-Optimized-mHRP-myc-mB7. pcDNA3.1-Renilla luciferase was a kind gift from Dr. Tian-Lu Cheng (Kaohsiung Medical University). pCMV-Cypridina Luc Vector was obtained from ThermoFisher (16,150, Waltham, US), by adding 5′HindIII and 3′SalI with PCR, Cypridina luciferase (CLuc, without stop codon) was subcloned into pLNCX-Optimized sHRP-myc vector (with myc-tag and stop codon) to generate pLNCX-CMV-CLuc-myc. pCMV-Cypridina Luc Vector underwent two subsequent PCR procedures to add SalI, myc tag and stop codon, and 5′NcoI and 3′FseI to subclone CLuc-myc to generate pTEAD1-CLuc-myc or pAP- 1-CLuc-myc. .. Forty-eight hours after transient transfection, 10 μL of supernatants were aspirated from cultured cells and mixed with 150 μL of ABTS (0.4 mg/mL, Sigma-Aldrich, Burlington, US) with 0.01% hydrogen peroxide (Sigma-Aldrich, Burlington, US) or TMB (0.1 mg/mL, 100 μL, Promega, Madison, US), and rotated at 50 rpm, at RT on a rotator.

    Article Title: FOSL1 Orchestrates Epigenetic Reprogramming of Anaplastic Thyroid Cancer and Suppresses Natural Killer Cell-Mediated Antitumor Immunity
    Article Snippet: 2 Anaplastic thyroid cancer (ATC) is the most aggressive type of thyroid cancer with 37 survival time of only 7-10 months.. Previous work revealed reduced proportions and 38 cytotoxicity of natural killer (NK) cells in ATC tumor microenvironment (TME).. Here, we 39 investigated the role of super-enhancers (SEs), clusters of adjacent enhancers and drive high 40 expression of genes, in reshaping the TME in ATC.

    Article Title: Evidence for G-quadruplex-mediated transactivation by the immediate-early 2 protein of human cytomegalovirus
    Article Snippet: .. To produce the UL146 promoter-driven luciferase reporter plasmid (UL146p-Luc), the UL146 promoter region (−927 to +108) was PCR-amplified as an NheI/NcoI fragment and cloned into pGL3-basic (Addgene). ..

    Article Title: Preclinical and clinical evaluation of intratumoral injection of an IL-12 expressing SKV-012 oncolytic virus for advanced solid tumors.
    Article Snippet: FlowJo and CytExpert software were used for cytometry data analyses. .. Survivin core promoter (−255 to 0) was cloned into the multiple cloning sites of pGL3- Basic (Addgene, Plasmid No 212936) upstream of the firefly luciferase gene with primers flanked with MluI or XhoI sites to create pGL3- Survivin. ..

    Polymerase Chain Reaction:

    Article Title: Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management
    Article Snippet: .. Briefly, signal peptide (SP)-HRP (secreted HRP (sHRP)) was cloned from pLNCX-Optimized mHRP-myc-mB7 vector, as described in our previous work [ ], by PCR with 5′HindIII and 3′ClaI flanking SP-HRP-myc and the transmembrane mB7 sequence was removed to obtain pLNCX-Optimized sHRP-myc. pTEAD1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc, removing mB7 and subcloning into 8xGTIIC-luciferase (Addgene plasmid #34,615). pAP- 1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc and subcloned into 3 × AP- 1 in pGL3-basic (pAP- 1-Luciferase, Addgene plasmid #40,342). pNF-κB-sHRP-myc was generated by PCR with 5′HindIII and 3′EcoRI flanking sHRP-myc from pLNCX-Optimized mHRP-myc-mB7 and subcloned into pGL4.32[luc2P/NF-κB-RE/Hygro] (E8491, Promega, Madison, US). cHRP was generated by PCR with 5′HindIII and 3′SalI to remove SP and subcloned into pLNCX-Optimized-mHRP-myc-mB7. pcDNA3.1-Renilla luciferase was a kind gift from Dr. Tian-Lu Cheng (Kaohsiung Medical University). pCMV-Cypridina Luc Vector was obtained from ThermoFisher (16,150, Waltham, US), by adding 5′HindIII and 3′SalI with PCR, Cypridina luciferase (CLuc, without stop codon) was subcloned into pLNCX-Optimized sHRP-myc vector (with myc-tag and stop codon) to generate pLNCX-CMV-CLuc-myc. pCMV-Cypridina Luc Vector underwent two subsequent PCR procedures to add SalI, myc tag and stop codon, and 5′NcoI and 3′FseI to subclone CLuc-myc to generate pTEAD1-CLuc-myc or pAP- 1-CLuc-myc. .. Forty-eight hours after transient transfection, 10 μL of supernatants were aspirated from cultured cells and mixed with 150 μL of ABTS (0.4 mg/mL, Sigma-Aldrich, Burlington, US) with 0.01% hydrogen peroxide (Sigma-Aldrich, Burlington, US) or TMB (0.1 mg/mL, 100 μL, Promega, Madison, US), and rotated at 50 rpm, at RT on a rotator.

    Article Title: Evidence for G-quadruplex-mediated transactivation by the immediate-early 2 protein of human cytomegalovirus
    Article Snippet: .. To produce the UL146 promoter-driven luciferase reporter plasmid (UL146p-Luc), the UL146 promoter region (−927 to +108) was PCR-amplified as an NheI/NcoI fragment and cloned into pGL3-basic (Addgene). ..

    Sequencing:

    Article Title: Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management
    Article Snippet: .. Briefly, signal peptide (SP)-HRP (secreted HRP (sHRP)) was cloned from pLNCX-Optimized mHRP-myc-mB7 vector, as described in our previous work [ ], by PCR with 5′HindIII and 3′ClaI flanking SP-HRP-myc and the transmembrane mB7 sequence was removed to obtain pLNCX-Optimized sHRP-myc. pTEAD1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc, removing mB7 and subcloning into 8xGTIIC-luciferase (Addgene plasmid #34,615). pAP- 1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc and subcloned into 3 × AP- 1 in pGL3-basic (pAP- 1-Luciferase, Addgene plasmid #40,342). pNF-κB-sHRP-myc was generated by PCR with 5′HindIII and 3′EcoRI flanking sHRP-myc from pLNCX-Optimized mHRP-myc-mB7 and subcloned into pGL4.32[luc2P/NF-κB-RE/Hygro] (E8491, Promega, Madison, US). cHRP was generated by PCR with 5′HindIII and 3′SalI to remove SP and subcloned into pLNCX-Optimized-mHRP-myc-mB7. pcDNA3.1-Renilla luciferase was a kind gift from Dr. Tian-Lu Cheng (Kaohsiung Medical University). pCMV-Cypridina Luc Vector was obtained from ThermoFisher (16,150, Waltham, US), by adding 5′HindIII and 3′SalI with PCR, Cypridina luciferase (CLuc, without stop codon) was subcloned into pLNCX-Optimized sHRP-myc vector (with myc-tag and stop codon) to generate pLNCX-CMV-CLuc-myc. pCMV-Cypridina Luc Vector underwent two subsequent PCR procedures to add SalI, myc tag and stop codon, and 5′NcoI and 3′FseI to subclone CLuc-myc to generate pTEAD1-CLuc-myc or pAP- 1-CLuc-myc. .. Forty-eight hours after transient transfection, 10 μL of supernatants were aspirated from cultured cells and mixed with 150 μL of ABTS (0.4 mg/mL, Sigma-Aldrich, Burlington, US) with 0.01% hydrogen peroxide (Sigma-Aldrich, Burlington, US) or TMB (0.1 mg/mL, 100 μL, Promega, Madison, US), and rotated at 50 rpm, at RT on a rotator.

    Generated:

    Article Title: Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management
    Article Snippet: .. Briefly, signal peptide (SP)-HRP (secreted HRP (sHRP)) was cloned from pLNCX-Optimized mHRP-myc-mB7 vector, as described in our previous work [ ], by PCR with 5′HindIII and 3′ClaI flanking SP-HRP-myc and the transmembrane mB7 sequence was removed to obtain pLNCX-Optimized sHRP-myc. pTEAD1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc, removing mB7 and subcloning into 8xGTIIC-luciferase (Addgene plasmid #34,615). pAP- 1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc and subcloned into 3 × AP- 1 in pGL3-basic (pAP- 1-Luciferase, Addgene plasmid #40,342). pNF-κB-sHRP-myc was generated by PCR with 5′HindIII and 3′EcoRI flanking sHRP-myc from pLNCX-Optimized mHRP-myc-mB7 and subcloned into pGL4.32[luc2P/NF-κB-RE/Hygro] (E8491, Promega, Madison, US). cHRP was generated by PCR with 5′HindIII and 3′SalI to remove SP and subcloned into pLNCX-Optimized-mHRP-myc-mB7. pcDNA3.1-Renilla luciferase was a kind gift from Dr. Tian-Lu Cheng (Kaohsiung Medical University). pCMV-Cypridina Luc Vector was obtained from ThermoFisher (16,150, Waltham, US), by adding 5′HindIII and 3′SalI with PCR, Cypridina luciferase (CLuc, without stop codon) was subcloned into pLNCX-Optimized sHRP-myc vector (with myc-tag and stop codon) to generate pLNCX-CMV-CLuc-myc. pCMV-Cypridina Luc Vector underwent two subsequent PCR procedures to add SalI, myc tag and stop codon, and 5′NcoI and 3′FseI to subclone CLuc-myc to generate pTEAD1-CLuc-myc or pAP- 1-CLuc-myc. .. Forty-eight hours after transient transfection, 10 μL of supernatants were aspirated from cultured cells and mixed with 150 μL of ABTS (0.4 mg/mL, Sigma-Aldrich, Burlington, US) with 0.01% hydrogen peroxide (Sigma-Aldrich, Burlington, US) or TMB (0.1 mg/mL, 100 μL, Promega, Madison, US), and rotated at 50 rpm, at RT on a rotator.

    Subcloning:

    Article Title: Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management
    Article Snippet: .. Briefly, signal peptide (SP)-HRP (secreted HRP (sHRP)) was cloned from pLNCX-Optimized mHRP-myc-mB7 vector, as described in our previous work [ ], by PCR with 5′HindIII and 3′ClaI flanking SP-HRP-myc and the transmembrane mB7 sequence was removed to obtain pLNCX-Optimized sHRP-myc. pTEAD1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc, removing mB7 and subcloning into 8xGTIIC-luciferase (Addgene plasmid #34,615). pAP- 1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc and subcloned into 3 × AP- 1 in pGL3-basic (pAP- 1-Luciferase, Addgene plasmid #40,342). pNF-κB-sHRP-myc was generated by PCR with 5′HindIII and 3′EcoRI flanking sHRP-myc from pLNCX-Optimized mHRP-myc-mB7 and subcloned into pGL4.32[luc2P/NF-κB-RE/Hygro] (E8491, Promega, Madison, US). cHRP was generated by PCR with 5′HindIII and 3′SalI to remove SP and subcloned into pLNCX-Optimized-mHRP-myc-mB7. pcDNA3.1-Renilla luciferase was a kind gift from Dr. Tian-Lu Cheng (Kaohsiung Medical University). pCMV-Cypridina Luc Vector was obtained from ThermoFisher (16,150, Waltham, US), by adding 5′HindIII and 3′SalI with PCR, Cypridina luciferase (CLuc, without stop codon) was subcloned into pLNCX-Optimized sHRP-myc vector (with myc-tag and stop codon) to generate pLNCX-CMV-CLuc-myc. pCMV-Cypridina Luc Vector underwent two subsequent PCR procedures to add SalI, myc tag and stop codon, and 5′NcoI and 3′FseI to subclone CLuc-myc to generate pTEAD1-CLuc-myc or pAP- 1-CLuc-myc. .. Forty-eight hours after transient transfection, 10 μL of supernatants were aspirated from cultured cells and mixed with 150 μL of ABTS (0.4 mg/mL, Sigma-Aldrich, Burlington, US) with 0.01% hydrogen peroxide (Sigma-Aldrich, Burlington, US) or TMB (0.1 mg/mL, 100 μL, Promega, Madison, US), and rotated at 50 rpm, at RT on a rotator.

    Plasmid Preparation:

    Article Title: Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management
    Article Snippet: .. Briefly, signal peptide (SP)-HRP (secreted HRP (sHRP)) was cloned from pLNCX-Optimized mHRP-myc-mB7 vector, as described in our previous work [ ], by PCR with 5′HindIII and 3′ClaI flanking SP-HRP-myc and the transmembrane mB7 sequence was removed to obtain pLNCX-Optimized sHRP-myc. pTEAD1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc, removing mB7 and subcloning into 8xGTIIC-luciferase (Addgene plasmid #34,615). pAP- 1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc and subcloned into 3 × AP- 1 in pGL3-basic (pAP- 1-Luciferase, Addgene plasmid #40,342). pNF-κB-sHRP-myc was generated by PCR with 5′HindIII and 3′EcoRI flanking sHRP-myc from pLNCX-Optimized mHRP-myc-mB7 and subcloned into pGL4.32[luc2P/NF-κB-RE/Hygro] (E8491, Promega, Madison, US). cHRP was generated by PCR with 5′HindIII and 3′SalI to remove SP and subcloned into pLNCX-Optimized-mHRP-myc-mB7. pcDNA3.1-Renilla luciferase was a kind gift from Dr. Tian-Lu Cheng (Kaohsiung Medical University). pCMV-Cypridina Luc Vector was obtained from ThermoFisher (16,150, Waltham, US), by adding 5′HindIII and 3′SalI with PCR, Cypridina luciferase (CLuc, without stop codon) was subcloned into pLNCX-Optimized sHRP-myc vector (with myc-tag and stop codon) to generate pLNCX-CMV-CLuc-myc. pCMV-Cypridina Luc Vector underwent two subsequent PCR procedures to add SalI, myc tag and stop codon, and 5′NcoI and 3′FseI to subclone CLuc-myc to generate pTEAD1-CLuc-myc or pAP- 1-CLuc-myc. .. Forty-eight hours after transient transfection, 10 μL of supernatants were aspirated from cultured cells and mixed with 150 μL of ABTS (0.4 mg/mL, Sigma-Aldrich, Burlington, US) with 0.01% hydrogen peroxide (Sigma-Aldrich, Burlington, US) or TMB (0.1 mg/mL, 100 μL, Promega, Madison, US), and rotated at 50 rpm, at RT on a rotator.

    Article Title: Evidence for G-quadruplex-mediated transactivation by the immediate-early 2 protein of human cytomegalovirus
    Article Snippet: .. To produce the UL146 promoter-driven luciferase reporter plasmid (UL146p-Luc), the UL146 promoter region (−927 to +108) was PCR-amplified as an NheI/NcoI fragment and cloned into pGL3-basic (Addgene). ..

    Article Title: Preclinical and clinical evaluation of intratumoral injection of an IL-12 expressing SKV-012 oncolytic virus for advanced solid tumors.
    Article Snippet: FlowJo and CytExpert software were used for cytometry data analyses. .. Survivin core promoter (−255 to 0) was cloned into the multiple cloning sites of pGL3- Basic (Addgene, Plasmid No 212936) upstream of the firefly luciferase gene with primers flanked with MluI or XhoI sites to create pGL3- Survivin. ..

    Luciferase:

    Article Title: Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management
    Article Snippet: .. Briefly, signal peptide (SP)-HRP (secreted HRP (sHRP)) was cloned from pLNCX-Optimized mHRP-myc-mB7 vector, as described in our previous work [ ], by PCR with 5′HindIII and 3′ClaI flanking SP-HRP-myc and the transmembrane mB7 sequence was removed to obtain pLNCX-Optimized sHRP-myc. pTEAD1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc, removing mB7 and subcloning into 8xGTIIC-luciferase (Addgene plasmid #34,615). pAP- 1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc and subcloned into 3 × AP- 1 in pGL3-basic (pAP- 1-Luciferase, Addgene plasmid #40,342). pNF-κB-sHRP-myc was generated by PCR with 5′HindIII and 3′EcoRI flanking sHRP-myc from pLNCX-Optimized mHRP-myc-mB7 and subcloned into pGL4.32[luc2P/NF-κB-RE/Hygro] (E8491, Promega, Madison, US). cHRP was generated by PCR with 5′HindIII and 3′SalI to remove SP and subcloned into pLNCX-Optimized-mHRP-myc-mB7. pcDNA3.1-Renilla luciferase was a kind gift from Dr. Tian-Lu Cheng (Kaohsiung Medical University). pCMV-Cypridina Luc Vector was obtained from ThermoFisher (16,150, Waltham, US), by adding 5′HindIII and 3′SalI with PCR, Cypridina luciferase (CLuc, without stop codon) was subcloned into pLNCX-Optimized sHRP-myc vector (with myc-tag and stop codon) to generate pLNCX-CMV-CLuc-myc. pCMV-Cypridina Luc Vector underwent two subsequent PCR procedures to add SalI, myc tag and stop codon, and 5′NcoI and 3′FseI to subclone CLuc-myc to generate pTEAD1-CLuc-myc or pAP- 1-CLuc-myc. .. Forty-eight hours after transient transfection, 10 μL of supernatants were aspirated from cultured cells and mixed with 150 μL of ABTS (0.4 mg/mL, Sigma-Aldrich, Burlington, US) with 0.01% hydrogen peroxide (Sigma-Aldrich, Burlington, US) or TMB (0.1 mg/mL, 100 μL, Promega, Madison, US), and rotated at 50 rpm, at RT on a rotator.

    Article Title: FOSL1 Orchestrates Epigenetic Reprogramming of Anaplastic Thyroid Cancer and Suppresses Natural Killer Cell-Mediated Antitumor Immunity
    Article Snippet: 2 Anaplastic thyroid cancer (ATC) is the most aggressive type of thyroid cancer with 37 survival time of only 7-10 months.. Previous work revealed reduced proportions and 38 cytotoxicity of natural killer (NK) cells in ATC tumor microenvironment (TME).. Here, we 39 investigated the role of super-enhancers (SEs), clusters of adjacent enhancers and drive high 40 expression of genes, in reshaping the TME in ATC.

    Article Title: Evidence for G-quadruplex-mediated transactivation by the immediate-early 2 protein of human cytomegalovirus
    Article Snippet: .. To produce the UL146 promoter-driven luciferase reporter plasmid (UL146p-Luc), the UL146 promoter region (−927 to +108) was PCR-amplified as an NheI/NcoI fragment and cloned into pGL3-basic (Addgene). ..

    Article Title: Preclinical and clinical evaluation of intratumoral injection of an IL-12 expressing SKV-012 oncolytic virus for advanced solid tumors.
    Article Snippet: FlowJo and CytExpert software were used for cytometry data analyses. .. Survivin core promoter (−255 to 0) was cloned into the multiple cloning sites of pGL3- Basic (Addgene, Plasmid No 212936) upstream of the firefly luciferase gene with primers flanked with MluI or XhoI sites to create pGL3- Survivin. ..

    Synthesized:

    Article Title: A Genome-Wide Synthetic Lethal Screen Identifies Spermidine Synthase as a Target to Enhance Erdafitinib Efficacy in FGFR-Mutant Bladder Cancer
    Article Snippet: MGH-U3 and SW780 cells were infected with lentivirus expressing sgRNAs targeting SRM , and stable cell lines were selected using puromycin (Invitrogen). .. The EGFR promoter reporter was designed and synthesized into the pGL3-Basic (RRID: Addgene_212936) vector by TSINGKE. ..

    other:

    Article Title: Genetically engineered secretory horseradish peroxidase is a sensitive, stable, and affordable non-lytic reporter gene system for real-time promoter activity management.
    Article Snippet: Briefly, signal peptide (SP)-HRP (secreted HRP (sHRP)) was cloned from pLNCX-Optimized mHRP-myc-mB7 vector, as described in our previous work [21], by PCR with 5′HindIII and 3′ClaI flanking SP-HRP-myc and the transmembrane mB7 sequence was removed to obtain pLNCX-Optimized sHRP-myc. pTEAD1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc, removing mB7 and subcloning into 8xGTIICluciferase (Addgene plasmid #34,615). pAP- 1-sHRP-myc was generated by PCR with 5′NcoI and 3′FseI flanking sHRP-myc and subcloned into 3 × AP- 1 in pGL3-basic (pAP- 1-Luciferase, Addgene plasmid #40,342). pNF-κBsHRP-myc was generated by PCR with 5′HindIII and 3′EcoRI flanking sHRP-myc from pLNCX-Optimized mHRP-myc-mB7 and subcloned into pGL4.32[luc2P/ NF-κB-RE/Hygro] (E8491, Promega, Madison, US). cHRP was generated by PCR with 5′HindIII and 3′SalI to remove SP and subcloned into pLNCX-OptimizedmHRP-myc-mB7. pcDNA3.1-Renilla luciferase was a kind gift from Dr. Tian-Lu Cheng (Kaohsiung Medical University). pCMV-Cypridina Luc Vector was obtained from ThermoFisher (16,150, Waltham, US), by adding 5′HindIII and 3′SalI with PCR, Cypridina luciferase (CLuc, without stop codon) was subcloned into pLNCXOptimized sHRP-myc vector (with myc-tag and stop codon) to generate pLNCX-CMV-CLuc-myc. pCMVCypridina Luc Vector underwent two subsequent PCR procedures to add SalI, myc tag and stop codon, and 5′NcoI and 3′FseI to subclone CLuc-myc to generate pTEAD1-CLuc-myc or pAP- 1-CLuc-myc. sHRP reporter gene chromogenic assay Forty-eight hours after transient transfection, 10 μL of supernatants were aspirated from cultured cells and mixed with 150 μL of ABTS (0.4 mg/mL, Sigma-Aldrich, Burlington, US) with 0.01% hydrogen peroxide (SigmaAldrich, Burlington, US) or TMB (0.1 mg/mL, 100 μL, Promega, Madison, US), and rotated at 50 rpm, at RT on a rotator.

    Electroporation:

    Article Title: Amyotrophic Lateral Sclerosis-associated 3′ UTR enhancer embedded within CAV1 risk gene
    Article Snippet: .. Electroporation of SH-SY5Y cells with pGL3-Promoter (and derivatives, Promega #E1761), pGL3-Basic (Addgene #212936), pGL3-Control (Addgene #212937), and 759_Renilla (derived from pGL4.70[hRluc], Promega #E6881) was performed using the Neon ® Transfection System (Thermo Fisher) according to the manufacturer’s instructions. .. For luciferase reporter assays, transfections were performed in DMEM without phenol red (Cytiva, SH30284.01) supplemented with 10% FBS.

    Derivative Assay:

    Article Title: Amyotrophic Lateral Sclerosis-associated 3′ UTR enhancer embedded within CAV1 risk gene
    Article Snippet: .. Electroporation of SH-SY5Y cells with pGL3-Promoter (and derivatives, Promega #E1761), pGL3-Basic (Addgene #212936), pGL3-Control (Addgene #212937), and 759_Renilla (derived from pGL4.70[hRluc], Promega #E6881) was performed using the Neon ® Transfection System (Thermo Fisher) according to the manufacturer’s instructions. .. For luciferase reporter assays, transfections were performed in DMEM without phenol red (Cytiva, SH30284.01) supplemented with 10% FBS.

    Transfection:

    Article Title: Amyotrophic Lateral Sclerosis-associated 3′ UTR enhancer embedded within CAV1 risk gene
    Article Snippet: .. Electroporation of SH-SY5Y cells with pGL3-Promoter (and derivatives, Promega #E1761), pGL3-Basic (Addgene #212936), pGL3-Control (Addgene #212937), and 759_Renilla (derived from pGL4.70[hRluc], Promega #E6881) was performed using the Neon ® Transfection System (Thermo Fisher) according to the manufacturer’s instructions. .. For luciferase reporter assays, transfections were performed in DMEM without phenol red (Cytiva, SH30284.01) supplemented with 10% FBS.

    Cloning:

    Article Title: Preclinical and clinical evaluation of intratumoral injection of an IL-12 expressing SKV-012 oncolytic virus for advanced solid tumors.
    Article Snippet: FlowJo and CytExpert software were used for cytometry data analyses. .. Survivin core promoter (−255 to 0) was cloned into the multiple cloning sites of pGL3- Basic (Addgene, Plasmid No 212936) upstream of the firefly luciferase gene with primers flanked with MluI or XhoI sites to create pGL3- Survivin. ..



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    Estrogen promotes HNRNPAB expression via SP1. (A) Immunoblot assessment of HNRNPAB in 2 human normal breast epithelial cell lines and 11 human BC cell lines. (B) CPTAC database analyzed the relative HNRNPAB and ERα protein level in 105 BC patients. P value was determined by Pearson correlation analysis. (C) The relative expression of TFF1 and HNRNPAB mRNAs after stimulated with 10 nM E 2 in estrogen-deprived MCF-7 cells for 48 h analyzed by qRT-PCR. (D) Immunoblot assessment of HNRNPAB expression in MCF-7 with or without 10 nM E 2 in estrogen-deprived MCF-7 cells for 72 h, or in short-term oestrogen deprivation (STED) MCF-7 cells for 7 days and parental cells. (E) The schematic of HNRNPAB promoter region sequence selected from the transcription start site of -2 kb~+100 bp. (F) MCF-7 cells were transfected with <t>pGL3</t> basic reporter vector containing HNRNPAB promoter, and pRL-TK reporter control vector containing luciferase activity, as well as control siRNA or different siRNA sequences of JUN , FOS , FOXA1 and SP1 . The relative fluorescence activity was measured. (G) CPTAC database analyzed the relative HNRNPAB and SP1 protein level in 68 ER+ BC patients. P value was determined by Pearson correlation analysis. (H) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector containing different regions of HNRNPAB promoter. The relative fluorescence activity was measured. (I) The schematic of three predicted conserved SP1 binding sites on the upstream -425 bp~+100 bp regions of the human HNRNPAB gene. Red boxes represent the predicted SP1 binding sites. (J) The DNA regions enriched by SP1 in ChIP assay were verified by PCR. (K) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector or containing “b” or “b” mutant regions. The relative fluorescence activity was measured. (L) The relative expression of SP1 and HNRNPAB mRNAs in MCF-7 cells bearing control or SP1 shRNAs analyzed by qRT-PCR. (M) Immunoblot assessment of SP1 and HNRNPAB expression in MCF-7 cells with or without SP1 shRNAs in the presence or absence of 10 nM E 2 for 48 h. Data was shown as mean ± S.D. from three independent experiments. Unpaired two-tailed Student's t test (C, H, K) and one-way ANOVA followed by Tukey's multiple comparisons test (F, L). ns, P >0.05; ***, P <0.001; ****, P <0.0001.
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    Estrogen promotes HNRNPAB expression via SP1. (A) Immunoblot assessment of HNRNPAB in 2 human normal breast epithelial cell lines and 11 human BC cell lines. (B) CPTAC database analyzed the relative HNRNPAB and ERα protein level in 105 BC patients. P value was determined by Pearson correlation analysis. (C) The relative expression of TFF1 and HNRNPAB mRNAs after stimulated with 10 nM E 2 in estrogen-deprived MCF-7 cells for 48 h analyzed by qRT-PCR. (D) Immunoblot assessment of HNRNPAB expression in MCF-7 with or without 10 nM E 2 in estrogen-deprived MCF-7 cells for 72 h, or in short-term oestrogen deprivation (STED) MCF-7 cells for 7 days and parental cells. (E) The schematic of HNRNPAB promoter region sequence selected from the transcription start site of -2 kb~+100 bp. (F) MCF-7 cells were transfected with pGL3 basic reporter vector containing HNRNPAB promoter, and pRL-TK reporter control vector containing luciferase activity, as well as control siRNA or different siRNA sequences of JUN , FOS , FOXA1 and SP1 . The relative fluorescence activity was measured. (G) CPTAC database analyzed the relative HNRNPAB and SP1 protein level in 68 ER+ BC patients. P value was determined by Pearson correlation analysis. (H) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector containing different regions of HNRNPAB promoter. The relative fluorescence activity was measured. (I) The schematic of three predicted conserved SP1 binding sites on the upstream -425 bp~+100 bp regions of the human HNRNPAB gene. Red boxes represent the predicted SP1 binding sites. (J) The DNA regions enriched by SP1 in ChIP assay were verified by PCR. (K) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector or containing “b” or “b” mutant regions. The relative fluorescence activity was measured. (L) The relative expression of SP1 and HNRNPAB mRNAs in MCF-7 cells bearing control or SP1 shRNAs analyzed by qRT-PCR. (M) Immunoblot assessment of SP1 and HNRNPAB expression in MCF-7 cells with or without SP1 shRNAs in the presence or absence of 10 nM E 2 for 48 h. Data was shown as mean ± S.D. from three independent experiments. Unpaired two-tailed Student's t test (C, H, K) and one-way ANOVA followed by Tukey's multiple comparisons test (F, L). ns, P >0.05; ***, P <0.001; ****, P <0.0001.

    Journal: International Journal of Biological Sciences

    Article Title: The Interaction of CircESR1 and HNRNPAB Regulates Cell Cycle Transition of Breast Cancer Cell

    doi: 10.7150/ijbs.126014

    Figure Lengend Snippet: Estrogen promotes HNRNPAB expression via SP1. (A) Immunoblot assessment of HNRNPAB in 2 human normal breast epithelial cell lines and 11 human BC cell lines. (B) CPTAC database analyzed the relative HNRNPAB and ERα protein level in 105 BC patients. P value was determined by Pearson correlation analysis. (C) The relative expression of TFF1 and HNRNPAB mRNAs after stimulated with 10 nM E 2 in estrogen-deprived MCF-7 cells for 48 h analyzed by qRT-PCR. (D) Immunoblot assessment of HNRNPAB expression in MCF-7 with or without 10 nM E 2 in estrogen-deprived MCF-7 cells for 72 h, or in short-term oestrogen deprivation (STED) MCF-7 cells for 7 days and parental cells. (E) The schematic of HNRNPAB promoter region sequence selected from the transcription start site of -2 kb~+100 bp. (F) MCF-7 cells were transfected with pGL3 basic reporter vector containing HNRNPAB promoter, and pRL-TK reporter control vector containing luciferase activity, as well as control siRNA or different siRNA sequences of JUN , FOS , FOXA1 and SP1 . The relative fluorescence activity was measured. (G) CPTAC database analyzed the relative HNRNPAB and SP1 protein level in 68 ER+ BC patients. P value was determined by Pearson correlation analysis. (H) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector containing different regions of HNRNPAB promoter. The relative fluorescence activity was measured. (I) The schematic of three predicted conserved SP1 binding sites on the upstream -425 bp~+100 bp regions of the human HNRNPAB gene. Red boxes represent the predicted SP1 binding sites. (J) The DNA regions enriched by SP1 in ChIP assay were verified by PCR. (K) 293T cells were transfected with control or SP1 vector, and pRL-TK reporter control vector containing luciferase activity, as well as pGL3 basic reporter vector or containing “b” or “b” mutant regions. The relative fluorescence activity was measured. (L) The relative expression of SP1 and HNRNPAB mRNAs in MCF-7 cells bearing control or SP1 shRNAs analyzed by qRT-PCR. (M) Immunoblot assessment of SP1 and HNRNPAB expression in MCF-7 cells with or without SP1 shRNAs in the presence or absence of 10 nM E 2 for 48 h. Data was shown as mean ± S.D. from three independent experiments. Unpaired two-tailed Student's t test (C, H, K) and one-way ANOVA followed by Tukey's multiple comparisons test (F, L). ns, P >0.05; ***, P <0.001; ****, P <0.0001.

    Article Snippet: For transcription factor mediated HNRNPAB expression, 0.2 μg pGL3 Basic luciferase reporter (RRID: Addgene_48743), 50 nmol JUN or FOS or FOXA1 or SP1 siRNAs and 0.02 μg pRL-TK plasmid (RRID: Addgene_11313) were transfected into cells using Lipofectamine 2000 (Invitrogen, Carlsbad, USA).

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Sequencing, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, Fluorescence, Binding Assay, Mutagenesis, Two Tailed Test