Structured Review

Promega pgl4 10 luc2
Compound 7 inhibits EJ-1 bladder carcinoma growth in immunodeficient NOG mice. (A) Whole-body bioluminescence imaging of tumor cells. Immunodeficient NOG mice were intraperitoneally (i.p.) inoculated with 1 × 10 6 EJ-1 cells transfected with <t>pGL4.10[luc2]</t> on day 0 and treated with 2 μg/0.1 mL compound 7 (97.1 μg/kg) or 0.1 mL of PBS i.p. on days 3 and 6. On day 7, 0.1 mL of 15 mg/mL VivoGlo™ luciferin (Xenogen, Alameda, CA, USA) was injected i.p. and the mice were placed in the specimen chamber and photon emissions transmitted from the mice were then measured. This treatment regimen was repeated 7 times and the images from day 28 are shown. Luciferase units are photons/second/mouse. (B) Inhibition of tumor growth by compound 7 in vivo. The growth of EJ-1 was monitored for weeks 2 through 7 as described in (A) and the average photon flux/sec for four to five mice is plotted ± SEM. * p
Pgl4 10 Luc2, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Price from $9.99 to $1999.99
pgl4 10 luc2 - by Bioz Stars, 2020-07
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Images

1) Product Images from "Targeting Cancer Cells with a Bisphosphonate Prodrug"

Article Title: Targeting Cancer Cells with a Bisphosphonate Prodrug

Journal: ChemMedChem

doi: 10.1002/cmdc.201600465

Compound 7 inhibits EJ-1 bladder carcinoma growth in immunodeficient NOG mice. (A) Whole-body bioluminescence imaging of tumor cells. Immunodeficient NOG mice were intraperitoneally (i.p.) inoculated with 1 × 10 6 EJ-1 cells transfected with pGL4.10[luc2] on day 0 and treated with 2 μg/0.1 mL compound 7 (97.1 μg/kg) or 0.1 mL of PBS i.p. on days 3 and 6. On day 7, 0.1 mL of 15 mg/mL VivoGlo™ luciferin (Xenogen, Alameda, CA, USA) was injected i.p. and the mice were placed in the specimen chamber and photon emissions transmitted from the mice were then measured. This treatment regimen was repeated 7 times and the images from day 28 are shown. Luciferase units are photons/second/mouse. (B) Inhibition of tumor growth by compound 7 in vivo. The growth of EJ-1 was monitored for weeks 2 through 7 as described in (A) and the average photon flux/sec for four to five mice is plotted ± SEM. * p
Figure Legend Snippet: Compound 7 inhibits EJ-1 bladder carcinoma growth in immunodeficient NOG mice. (A) Whole-body bioluminescence imaging of tumor cells. Immunodeficient NOG mice were intraperitoneally (i.p.) inoculated with 1 × 10 6 EJ-1 cells transfected with pGL4.10[luc2] on day 0 and treated with 2 μg/0.1 mL compound 7 (97.1 μg/kg) or 0.1 mL of PBS i.p. on days 3 and 6. On day 7, 0.1 mL of 15 mg/mL VivoGlo™ luciferin (Xenogen, Alameda, CA, USA) was injected i.p. and the mice were placed in the specimen chamber and photon emissions transmitted from the mice were then measured. This treatment regimen was repeated 7 times and the images from day 28 are shown. Luciferase units are photons/second/mouse. (B) Inhibition of tumor growth by compound 7 in vivo. The growth of EJ-1 was monitored for weeks 2 through 7 as described in (A) and the average photon flux/sec for four to five mice is plotted ± SEM. * p

Techniques Used: Mouse Assay, Imaging, Transfection, Injection, Luciferase, Inhibition, In Vivo, Size-exclusion Chromatography

Related Articles

Clone Assay:

Article Title: Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis
Article Snippet: .. Human ERG cDNA (NCBI Accession NM_182918) was cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). pGL4.10[luc2] (Promega, Madison, USA) Firefly Luciferase empty vector, lacking a promoter sequence, was used as a control. pGL4.73[hRluc/SV40] (Promega) Renilla luciferase vector was used as an internal normalisation control in the luciferase assay. .. HUVEC were transfected with Genejuice transfection reagent (Merck Chemicals), as recommended.

Transfection:

Article Title: Targeting Cancer Cells with a Bisphosphonate Prodrug
Article Snippet: .. EJ-1 tumor cells (1 × 106 ) stably transfected with pGL4.10[luc2] (Promega, Madison, WI, USA) were intraperitoneally (i.p.) inoculated into immunodeficient NOG mice (obtained from the Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan). .. Mice were treated i.p. with 2 μg of compound 7 in 0.1 mL of PBS (97.1 μg/kg body weight for mice weighing 20.6 g), on day 3 and 6.

Amplification:

Article Title: miR-1199-5p and Zeb1 function in a double-negative feedback loop potentially coordinating EMT and tumour metastasis
Article Snippet: .. The PCR amplicon was digested with XhoI and HindIII and subcloned into the pGL4.10[luc2] Firefly luciferase reporter vector purchased from Promega. .. Using the QuikChange II site-directed mutagenesis Kit (Agilent), four different miR-1199/2210011C24Rik promoter luciferase mutants exhibiting point mutations in the Zeb1 E-box-binding motifs (wt: CAGGTG; mut: CATTTG) at positions −18, −133, −507 and −584 bp from the TSS were generated from the original miR-1199/2210011C24Rik promoter reporter.

Stable Transfection:

Article Title: Targeting Cancer Cells with a Bisphosphonate Prodrug
Article Snippet: .. EJ-1 tumor cells (1 × 106 ) stably transfected with pGL4.10[luc2] (Promega, Madison, WI, USA) were intraperitoneally (i.p.) inoculated into immunodeficient NOG mice (obtained from the Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan). .. Mice were treated i.p. with 2 μg of compound 7 in 0.1 mL of PBS (97.1 μg/kg body weight for mice weighing 20.6 g), on day 3 and 6.

Polymerase Chain Reaction:

Article Title: miR-1199-5p and Zeb1 function in a double-negative feedback loop potentially coordinating EMT and tumour metastasis
Article Snippet: .. The PCR amplicon was digested with XhoI and HindIII and subcloned into the pGL4.10[luc2] Firefly luciferase reporter vector purchased from Promega. .. Using the QuikChange II site-directed mutagenesis Kit (Agilent), four different miR-1199/2210011C24Rik promoter luciferase mutants exhibiting point mutations in the Zeb1 E-box-binding motifs (wt: CAGGTG; mut: CATTTG) at positions −18, −133, −507 and −584 bp from the TSS were generated from the original miR-1199/2210011C24Rik promoter reporter.

Article Title: Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products
Article Snippet: .. The ORFs for the synthetic firefly luciferase gene (luc2 ), HaloTag, and Venus were obtained from pGL4.10[luc2 ], pFC8A vector (Promega), and (EYFP-F46L/V68L/M153T/V163A/S175G)/pCS2 kindly provided by Dr. Atsushi Miyawaki (Brain Science Institute, RIKEN), respectively, by PCR and were subcloned into the pF1KT7 Flexi vector. .. Each ORF was transferred to seven different kinds of modified pTD2 expression vectors whose cloning sites were flanked at both sides with specified sequences.

Article Title: Transcriptional Activation of Low-Density Lipoprotein Receptor Gene by DJ-1 and Effect of DJ-1 on Cholesterol Homeostasis
Article Snippet: .. PCR products were digested with HindIII and NcoI and inserted into HindIII and NcoI sites of pGL4.10[luc2] (Promega, Madison, WI, USA). .. NIH3T3 and D2 cells in 24-well dishes were transfected with 0.75 µg of pGL4.10-hLDLR 200 or its deletion reporter plasmids and various amounts (0–1.0 µg) of pEF-DJ-1-HA together with 0.25 µg of pCMV-β-gal by the calcium phosphate method .

Construct:

Article Title: Cap-independent translation through the p27 5?-UTR
Article Snippet: .. Promoterless bicistronic constructs, pGL4.10-F/R-472 and pGL4.10-F/R-472R, were prepared by ligating the NheI/XbaI fragments from pGL4.70[hRluc]-472 and pGL4.70[hRluc]-472R into the XbaI site of pGL4.10[luc2]. .. Cell culture, DNA transfections and reporter gene assays Breast cancer cell lines were grown in Dulbecco's modified Eagle Medium (DMEM) containing 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin.

Mouse Assay:

Article Title: Targeting Cancer Cells with a Bisphosphonate Prodrug
Article Snippet: .. EJ-1 tumor cells (1 × 106 ) stably transfected with pGL4.10[luc2] (Promega, Madison, WI, USA) were intraperitoneally (i.p.) inoculated into immunodeficient NOG mice (obtained from the Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan). .. Mice were treated i.p. with 2 μg of compound 7 in 0.1 mL of PBS (97.1 μg/kg body weight for mice weighing 20.6 g), on day 3 and 6.

Modification:

Article Title: Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns
Article Snippet: .. Inserts were partially sequenced to confirm identity, then were moved into the BamHI site, downstream from the firefly luciferase gene, of pGL4.10[luc2] (Promega) that had been modified to contain a Sox9 promoter (∼530 bp) at the HindIII site. .. DLD1 cells were transfected with reporter constructs using Lipofectamine 2000 (Invitrogen).

Luciferase:

Article Title: miR-1199-5p and Zeb1 function in a double-negative feedback loop potentially coordinating EMT and tumour metastasis
Article Snippet: .. The PCR amplicon was digested with XhoI and HindIII and subcloned into the pGL4.10[luc2] Firefly luciferase reporter vector purchased from Promega. .. Using the QuikChange II site-directed mutagenesis Kit (Agilent), four different miR-1199/2210011C24Rik promoter luciferase mutants exhibiting point mutations in the Zeb1 E-box-binding motifs (wt: CAGGTG; mut: CATTTG) at positions −18, −133, −507 and −584 bp from the TSS were generated from the original miR-1199/2210011C24Rik promoter reporter.

Article Title: Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis
Article Snippet: .. Human ERG cDNA (NCBI Accession NM_182918) was cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). pGL4.10[luc2] (Promega, Madison, USA) Firefly Luciferase empty vector, lacking a promoter sequence, was used as a control. pGL4.73[hRluc/SV40] (Promega) Renilla luciferase vector was used as an internal normalisation control in the luciferase assay. .. HUVEC were transfected with Genejuice transfection reagent (Merck Chemicals), as recommended.

Article Title: Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products
Article Snippet: .. The ORFs for the synthetic firefly luciferase gene (luc2 ), HaloTag, and Venus were obtained from pGL4.10[luc2 ], pFC8A vector (Promega), and (EYFP-F46L/V68L/M153T/V163A/S175G)/pCS2 kindly provided by Dr. Atsushi Miyawaki (Brain Science Institute, RIKEN), respectively, by PCR and were subcloned into the pF1KT7 Flexi vector. .. Each ORF was transferred to seven different kinds of modified pTD2 expression vectors whose cloning sites were flanked at both sides with specified sequences.

Article Title: Genomic distribution of CHD7 on chromatin tracks H3K4 methylation patterns
Article Snippet: .. Inserts were partially sequenced to confirm identity, then were moved into the BamHI site, downstream from the firefly luciferase gene, of pGL4.10[luc2] (Promega) that had been modified to contain a Sox9 promoter (∼530 bp) at the HindIII site. .. DLD1 cells were transfected with reporter constructs using Lipofectamine 2000 (Invitrogen).

Expressing:

Article Title: Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis
Article Snippet: .. Human ERG cDNA (NCBI Accession NM_182918) was cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). pGL4.10[luc2] (Promega, Madison, USA) Firefly Luciferase empty vector, lacking a promoter sequence, was used as a control. pGL4.73[hRluc/SV40] (Promega) Renilla luciferase vector was used as an internal normalisation control in the luciferase assay. .. HUVEC were transfected with Genejuice transfection reagent (Merck Chemicals), as recommended.

Sequencing:

Article Title: Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis
Article Snippet: .. Human ERG cDNA (NCBI Accession NM_182918) was cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). pGL4.10[luc2] (Promega, Madison, USA) Firefly Luciferase empty vector, lacking a promoter sequence, was used as a control. pGL4.73[hRluc/SV40] (Promega) Renilla luciferase vector was used as an internal normalisation control in the luciferase assay. .. HUVEC were transfected with Genejuice transfection reagent (Merck Chemicals), as recommended.

Plasmid Preparation:

Article Title: miR-1199-5p and Zeb1 function in a double-negative feedback loop potentially coordinating EMT and tumour metastasis
Article Snippet: .. The PCR amplicon was digested with XhoI and HindIII and subcloned into the pGL4.10[luc2] Firefly luciferase reporter vector purchased from Promega. .. Using the QuikChange II site-directed mutagenesis Kit (Agilent), four different miR-1199/2210011C24Rik promoter luciferase mutants exhibiting point mutations in the Zeb1 E-box-binding motifs (wt: CAGGTG; mut: CATTTG) at positions −18, −133, −507 and −584 bp from the TSS were generated from the original miR-1199/2210011C24Rik promoter reporter.

Article Title: Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis
Article Snippet: .. Human ERG cDNA (NCBI Accession NM_182918) was cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). pGL4.10[luc2] (Promega, Madison, USA) Firefly Luciferase empty vector, lacking a promoter sequence, was used as a control. pGL4.73[hRluc/SV40] (Promega) Renilla luciferase vector was used as an internal normalisation control in the luciferase assay. .. HUVEC were transfected with Genejuice transfection reagent (Merck Chemicals), as recommended.

Article Title: Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products
Article Snippet: .. The ORFs for the synthetic firefly luciferase gene (luc2 ), HaloTag, and Venus were obtained from pGL4.10[luc2 ], pFC8A vector (Promega), and (EYFP-F46L/V68L/M153T/V163A/S175G)/pCS2 kindly provided by Dr. Atsushi Miyawaki (Brain Science Institute, RIKEN), respectively, by PCR and were subcloned into the pF1KT7 Flexi vector. .. Each ORF was transferred to seven different kinds of modified pTD2 expression vectors whose cloning sites were flanked at both sides with specified sequences.

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    Promega il 17c pgl4 10 luc2 reporter vector
    <t>IL-17C</t> promoter activation by TNFα is mediated by a NF-κB-dependent mechanism. Cultured normal human keratinocytes were transfected with wild-type <t>(IL-17C-2-3204-luc2)</t> IL-17C-promoter-luciferase plasmids together with an internal control
    Il 17c Pgl4 10 Luc2 Reporter Vector, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/il 17c pgl4 10 luc2 reporter vector/product/Promega
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    il 17c pgl4 10 luc2 reporter vector - by Bioz Stars, 2020-07
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    Promega pgl4 10
    Reporter assay for evaluation of the promoter activities of Cdc6 , Dele -F, and Dele -R G4 DNAs. The G4-forming sequences were cloned into the <t>pGL4.10</t> vector not containing any promoter. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activities relative to the pGL4.10 vector are shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: ***P
    Pgl4 10, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Promega pgl4 basic
    PARG knockdown leads to downregulation of PARP1 gene expression and its promoter activity. (A) HeLa S3 cells were transfected with 100 nM of the PARG -siRNA. After 48 h of incubation, total RNAs were isolated and subjected to real-time qPCR analysis with PARP1 - or GAPDH -specific primer pairs. Histograms show relative expression of the PARP1 gene compared to the GAPDH gene. The data are shown as the means ± SEM of three independent experiments. (B) HeLa S3 cells were transfected with 100 nM of the PARG -siRNA. After 48 h of incubation, cells were transfected with Luc-reporter plasmids, <t>pGL4-basic</t> (bars 1 and 2), pGL4-PARP1 (bars 3 and 4), and pGL4-KBSTΔ6 (bars 5 and 6). After a further 24 h of incubation, cells were harvested and a dual Luc assay was carried out. (C) Similar experiments were carried out by transfecting pGL4-basic (bars 1 and 2), pGL4-PARP1Δ1 (bars 3 and 4), and pGL4-PARP1Δ2 (bars 5 and 6). (B and C) Histograms show the relative Luc activities compared to cells transfected with the pGL3-promoter vector. The data are shown as the means + SEM of three independent experiments.
    Pgl4 Basic, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IL-17C promoter activation by TNFα is mediated by a NF-κB-dependent mechanism. Cultured normal human keratinocytes were transfected with wild-type (IL-17C-2-3204-luc2) IL-17C-promoter-luciferase plasmids together with an internal control

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor Necrosis Factor ?-Mediated Induction of Interleukin 17C in Human Keratinocytes Is Controlled by Nuclear Factor ?B

    doi: 10.1074/jbc.M111.240671

    Figure Lengend Snippet: IL-17C promoter activation by TNFα is mediated by a NF-κB-dependent mechanism. Cultured normal human keratinocytes were transfected with wild-type (IL-17C-2-3204-luc2) IL-17C-promoter-luciferase plasmids together with an internal control

    Article Snippet: 24 h after transfection, cells were stimulated with TNFα for 24 h. After stimulation, cells were harvested with 100 μl of passive lysis buffer from Promega, and firefly luciferase activity from the IL-17C-pGL4.10[luc2] reporter vector and Renilla luciferase activity were measured by the Dual Luciferase assay system (Promega) on a Fluoroskan Ascent Fl (BIE & Berntsen, Rodovre, Denmark).

    Techniques: Activation Assay, Cell Culture, Transfection, Luciferase

    Reporter assay for evaluation of the promoter activities of Cdc6 , Dele -F, and Dele -R G4 DNAs. The G4-forming sequences were cloned into the pGL4.10 vector not containing any promoter. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activities relative to the pGL4.10 vector are shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: ***P

    Journal: BMC Molecular Biology

    Article Title: Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands

    doi: 10.1186/s12867-017-0094-z

    Figure Lengend Snippet: Reporter assay for evaluation of the promoter activities of Cdc6 , Dele -F, and Dele -R G4 DNAs. The G4-forming sequences were cloned into the pGL4.10 vector not containing any promoter. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activities relative to the pGL4.10 vector are shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: ***P

    Article Snippet: Plasmid construction The G4 DNAs and mutant-type DNAs (Additional file ) were cloned in the Sfi I site of the pGL4.23 [luc2/minP] or pGL4.10 [luc2] (Promega Corporation, Madison, WI, USA) and then transformed into E. coli DH5α (TOYOBO, Osaka, Japan).

    Techniques: Reporter Assay, Clone Assay, Plasmid Preparation, Mutagenesis, Luciferase

    Reporter assay for evaluation of the transcriptional activity of Cdc6 , Dele -F, and Dele -R G4 DNAs in CGI sequences. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activity relative to the pGL4.10 is shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: *P

    Journal: BMC Molecular Biology

    Article Title: Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands

    doi: 10.1186/s12867-017-0094-z

    Figure Lengend Snippet: Reporter assay for evaluation of the transcriptional activity of Cdc6 , Dele -F, and Dele -R G4 DNAs in CGI sequences. Black bars represent the wild-types and white bars represent the mutant-types. Luciferase activity relative to the pGL4.10 is shown (mean ± SD, n = 3). Wild- and mutant-types samples t-test differences: *P

    Article Snippet: Plasmid construction The G4 DNAs and mutant-type DNAs (Additional file ) were cloned in the Sfi I site of the pGL4.23 [luc2/minP] or pGL4.10 [luc2] (Promega Corporation, Madison, WI, USA) and then transformed into E. coli DH5α (TOYOBO, Osaka, Japan).

    Techniques: Reporter Assay, Activity Assay, Mutagenesis, Luciferase

    PIK3IP1 promoter is inactivated by Ras. a mRNA abundance of PIK3IP1 in BJ-H-RasV12-ER and N4-H-RasV12-ER cells with or without 4-HT treatment was assessed by RT-qPCR. PIK3IP1 expression without 4-HT (Black bar) was designated as 1, and the value with 4-HT treatment (white bar) was normalized to this. b Dissection of the Ras-regulated PIK3IP1 promoter. The indicated genomic fragments starting 4500 bp upstream of the PIK3IP1 transcriptional start site were cloned into pGL4.10 Empty vector (EV) served as a negative control (EV). Reporter constructs were co-transfected with either control or K-Ras expression vector. Open boxes correspond to the promoter region upstream of the transcription start site. c Comparison of PIK3IP1 transcriptional activity in PIK3IP1 promoter constructs and promoter construct with the distal enhancer. Data were presented as the means ± SD of three independent experiments. ** p

    Journal: Oncogenesis

    Article Title: A Ras-LSD1 axis activates PI3K signaling through PIK3IP1 suppression

    doi: 10.1038/s41389-019-0185-4

    Figure Lengend Snippet: PIK3IP1 promoter is inactivated by Ras. a mRNA abundance of PIK3IP1 in BJ-H-RasV12-ER and N4-H-RasV12-ER cells with or without 4-HT treatment was assessed by RT-qPCR. PIK3IP1 expression without 4-HT (Black bar) was designated as 1, and the value with 4-HT treatment (white bar) was normalized to this. b Dissection of the Ras-regulated PIK3IP1 promoter. The indicated genomic fragments starting 4500 bp upstream of the PIK3IP1 transcriptional start site were cloned into pGL4.10 Empty vector (EV) served as a negative control (EV). Reporter constructs were co-transfected with either control or K-Ras expression vector. Open boxes correspond to the promoter region upstream of the transcription start site. c Comparison of PIK3IP1 transcriptional activity in PIK3IP1 promoter constructs and promoter construct with the distal enhancer. Data were presented as the means ± SD of three independent experiments. ** p

    Article Snippet: All PCR products were digested with SacI and XhoI and cloned into pGL4.10 [luc2] (Promega).

    Techniques: Quantitative RT-PCR, Expressing, Dissection, Clone Assay, Plasmid Preparation, Negative Control, Construct, Transfection, Activity Assay

    Ras activation using 4-HT increases LSD1 on PIK3IP1 enhancer and promoter regions of both BJ-H-RasV12-ER and N4-H-RasV12-ER cells. a Pharmacological inhibition of LSD1 with S2101 promoted PIK3IP1 gene expression in multiple Ras/Raf-mutant cancer cell lines. PIK3IP1 mRNA abundance in each cell line without treatment (black bars) was set to 1. b LSD1 binding to the promoter and enhancer of PIK3IP1 was enhanced by Ras activation. BJ-H-RasV12-ER and N4-H-RasV12-ER cells were treated with 4-HT for 24 h and then examined by LSD1 ChIP-qPCR. LSD1 without 4-HT (Black bar) was set to 1. c LSD1 knockdown increased activating marks at the PIK3IP1 enhancer. ChIP-qPCR analysis of indicated histone marks was performed in BJ-H-RasV12-ER and N4-H-RasV12-ER cells after LSD 1 knockdown. d PI3KIP1 promoter activity regulated by inhibitors of MEK, ERK, and LSD1. 293 T cells were co-transfected with pGL4.10-Luc (−250/−100), minimal regulatory region of promoter, and either K-Ras expression or K-Ras empty plasmids. Twenty hour after transfection, K-Ras co-transfected cells were treated with the indicated compounds. After 8 h, the cells were collected and assayed for luciferase activity. Values were normalized against luciferase activity of DMSO treated cells co-transfected with K-Ras empty plasmids. The experiments were performed in triplicate. e Relative PIK3IP1 mRNA expression by qPCR analysis in BJ-H-RasV12-ER cells were treated the indicated compounds with or without 4-HT for 24 h. PIK3IP1 expression without 4-HT was set to 1. Inhibitors and their concentrations used in d and e : LSD1 inhibitor (S2101, 20 μM), MEK inhibitor (U0126, 10 μM), and ERK inhibitor (SCH772984, 1 μM). Data were presented as the means ± SD of three independent experiments. ** p

    Journal: Oncogenesis

    Article Title: A Ras-LSD1 axis activates PI3K signaling through PIK3IP1 suppression

    doi: 10.1038/s41389-019-0185-4

    Figure Lengend Snippet: Ras activation using 4-HT increases LSD1 on PIK3IP1 enhancer and promoter regions of both BJ-H-RasV12-ER and N4-H-RasV12-ER cells. a Pharmacological inhibition of LSD1 with S2101 promoted PIK3IP1 gene expression in multiple Ras/Raf-mutant cancer cell lines. PIK3IP1 mRNA abundance in each cell line without treatment (black bars) was set to 1. b LSD1 binding to the promoter and enhancer of PIK3IP1 was enhanced by Ras activation. BJ-H-RasV12-ER and N4-H-RasV12-ER cells were treated with 4-HT for 24 h and then examined by LSD1 ChIP-qPCR. LSD1 without 4-HT (Black bar) was set to 1. c LSD1 knockdown increased activating marks at the PIK3IP1 enhancer. ChIP-qPCR analysis of indicated histone marks was performed in BJ-H-RasV12-ER and N4-H-RasV12-ER cells after LSD 1 knockdown. d PI3KIP1 promoter activity regulated by inhibitors of MEK, ERK, and LSD1. 293 T cells were co-transfected with pGL4.10-Luc (−250/−100), minimal regulatory region of promoter, and either K-Ras expression or K-Ras empty plasmids. Twenty hour after transfection, K-Ras co-transfected cells were treated with the indicated compounds. After 8 h, the cells were collected and assayed for luciferase activity. Values were normalized against luciferase activity of DMSO treated cells co-transfected with K-Ras empty plasmids. The experiments were performed in triplicate. e Relative PIK3IP1 mRNA expression by qPCR analysis in BJ-H-RasV12-ER cells were treated the indicated compounds with or without 4-HT for 24 h. PIK3IP1 expression without 4-HT was set to 1. Inhibitors and their concentrations used in d and e : LSD1 inhibitor (S2101, 20 μM), MEK inhibitor (U0126, 10 μM), and ERK inhibitor (SCH772984, 1 μM). Data were presented as the means ± SD of three independent experiments. ** p

    Article Snippet: All PCR products were digested with SacI and XhoI and cloned into pGL4.10 [luc2] (Promega).

    Techniques: Activation Assay, Inhibition, Expressing, Mutagenesis, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Activity Assay, Transfection, Luciferase

    PARG knockdown leads to downregulation of PARP1 gene expression and its promoter activity. (A) HeLa S3 cells were transfected with 100 nM of the PARG -siRNA. After 48 h of incubation, total RNAs were isolated and subjected to real-time qPCR analysis with PARP1 - or GAPDH -specific primer pairs. Histograms show relative expression of the PARP1 gene compared to the GAPDH gene. The data are shown as the means ± SEM of three independent experiments. (B) HeLa S3 cells were transfected with 100 nM of the PARG -siRNA. After 48 h of incubation, cells were transfected with Luc-reporter plasmids, pGL4-basic (bars 1 and 2), pGL4-PARP1 (bars 3 and 4), and pGL4-KBSTΔ6 (bars 5 and 6). After a further 24 h of incubation, cells were harvested and a dual Luc assay was carried out. (C) Similar experiments were carried out by transfecting pGL4-basic (bars 1 and 2), pGL4-PARP1Δ1 (bars 3 and 4), and pGL4-PARP1Δ2 (bars 5 and 6). (B and C) Histograms show the relative Luc activities compared to cells transfected with the pGL3-promoter vector. The data are shown as the means + SEM of three independent experiments.

    Journal: Oncology Reports

    Article Title: PARP1 gene expression is downregulated by knockdown of PARG gene

    doi: 10.3892/or.2013.2321

    Figure Lengend Snippet: PARG knockdown leads to downregulation of PARP1 gene expression and its promoter activity. (A) HeLa S3 cells were transfected with 100 nM of the PARG -siRNA. After 48 h of incubation, total RNAs were isolated and subjected to real-time qPCR analysis with PARP1 - or GAPDH -specific primer pairs. Histograms show relative expression of the PARP1 gene compared to the GAPDH gene. The data are shown as the means ± SEM of three independent experiments. (B) HeLa S3 cells were transfected with 100 nM of the PARG -siRNA. After 48 h of incubation, cells were transfected with Luc-reporter plasmids, pGL4-basic (bars 1 and 2), pGL4-PARP1 (bars 3 and 4), and pGL4-KBSTΔ6 (bars 5 and 6). After a further 24 h of incubation, cells were harvested and a dual Luc assay was carried out. (C) Similar experiments were carried out by transfecting pGL4-basic (bars 1 and 2), pGL4-PARP1Δ1 (bars 3 and 4), and pGL4-PARP1Δ2 (bars 5 and 6). (B and C) Histograms show the relative Luc activities compared to cells transfected with the pGL3-promoter vector. The data are shown as the means + SEM of three independent experiments.

    Article Snippet: The amplified DNA fragments were digested with Kpn I and Xho I and ligated into the MCS of the pGL4-basic (pGL4.10[luc2]), vector (Promega) to make pGL4-PARP1.

    Techniques: Expressing, Activity Assay, Transfection, Incubation, Isolation, Real-time Polymerase Chain Reaction, Plasmid Preparation