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  • 90
    Thermo Fisher ptripz
    Intracranial (i.c.) xenograft formation assay of U251-NS Kaplan-Meier survival curves were was plotted from overall survivals of mice following i.c. implantation of U251-NS infectants of <t>pTRIPZ-lentiviral</t> constructs of vector, wild-type and variants of <t>EFEMP1.</t>
    Ptripz, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 557 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa ptripz
    Intracranial (i.c.) xenograft formation assay of U251-NS Kaplan-Meier survival curves were was plotted from overall survivals of mice following i.c. implantation of U251-NS infectants of <t>pTRIPZ-lentiviral</t> constructs of vector, wild-type and variants of <t>EFEMP1.</t>
    Ptripz, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Horizon Discovery ptripz
    Schematic representation of control forward <t>(pTRIPZ)</t> and novel reverse orientation (pTREX) lentiviral expression vectors. (A) The forward orientation lentiviral vector pTRIPZ is commercially available (GE Healthcare/Dharmacon). pTRIPZ allows expression of a transgene cloned between unique AgeI and ClaI sites and miRNAs cloned between unique XhoI and EcoRI sites present in the transgene 3′UTR. Both are transcribed from a <t>tet-inducible</t> minimal CMV promoter (TetO/CMV). A 3′ Ubiquitin C promoter (UBC) drives the constitutive expression of the reverse tetracycline transactivator 3 (rtTA3) protein as well as a puromycin (Puro) selectable marker located 3′ to an internal ribosome entry site (IRES). (B) The pTREX lentiviral expression vector was generated from pTRIPZ by inverting sequences encompassing the TetO/CMV and inserting an intron derived from the rat preproinsulin II gene 5′ of the inserted transgene. pTREX also contains a poly(A) addition site located 3′ to the inserted transgene. Unique XhoI and EcoRI sites located in the intron are used for miRNA expression. Transgenes are cloned between the unique AgeI and XbaI sites. Additionally, both vectors contain elements necessary for the HIV-1 life cycle including the long-terminal repeats (LTRs), ψ packaging signal, Rev response element (RRE) and the central polypurine tract (cPPT) as well as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which enhances vector expression. The 3′ LTR bears a large deletion in U3 rendering the lentivirus self-inactivating (SIN).
    Ptripz, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 92/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ptripz - by Bioz Stars, 2020-05
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    90
    GE Healthcare ptripz
    Schematic representation of control forward <t>(pTRIPZ)</t> and novel reverse orientation (pTREX) lentiviral expression vectors. (A) The forward orientation lentiviral vector pTRIPZ is commercially available (GE Healthcare/Dharmacon). pTRIPZ allows expression of a transgene cloned between unique AgeI and ClaI sites and miRNAs cloned between unique XhoI and EcoRI sites present in the transgene 3′UTR. Both are transcribed from a <t>tet-inducible</t> minimal CMV promoter (TetO/CMV). A 3′ Ubiquitin C promoter (UBC) drives the constitutive expression of the reverse tetracycline transactivator 3 (rtTA3) protein as well as a puromycin (Puro) selectable marker located 3′ to an internal ribosome entry site (IRES). (B) The pTREX lentiviral expression vector was generated from pTRIPZ by inverting sequences encompassing the TetO/CMV and inserting an intron derived from the rat preproinsulin II gene 5′ of the inserted transgene. pTREX also contains a poly(A) addition site located 3′ to the inserted transgene. Unique XhoI and EcoRI sites located in the intron are used for miRNA expression. Transgenes are cloned between the unique AgeI and XbaI sites. Additionally, both vectors contain elements necessary for the HIV-1 life cycle including the long-terminal repeats (LTRs), ψ packaging signal, Rev response element (RRE) and the central polypurine tract (cPPT) as well as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which enhances vector expression. The 3′ LTR bears a large deletion in U3 rendering the lentivirus self-inactivating (SIN).
    Ptripz, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 37 article reviews
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    ptripz - by Bioz Stars, 2020-05
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    90
    Thermo Fisher ptripz xrn1
    Schematic representation of control forward <t>(pTRIPZ)</t> and novel reverse orientation (pTREX) lentiviral expression vectors. (A) The forward orientation lentiviral vector pTRIPZ is commercially available (GE Healthcare/Dharmacon). pTRIPZ allows expression of a transgene cloned between unique AgeI and ClaI sites and miRNAs cloned between unique XhoI and EcoRI sites present in the transgene 3′UTR. Both are transcribed from a <t>tet-inducible</t> minimal CMV promoter (TetO/CMV). A 3′ Ubiquitin C promoter (UBC) drives the constitutive expression of the reverse tetracycline transactivator 3 (rtTA3) protein as well as a puromycin (Puro) selectable marker located 3′ to an internal ribosome entry site (IRES). (B) The pTREX lentiviral expression vector was generated from pTRIPZ by inverting sequences encompassing the TetO/CMV and inserting an intron derived from the rat preproinsulin II gene 5′ of the inserted transgene. pTREX also contains a poly(A) addition site located 3′ to the inserted transgene. Unique XhoI and EcoRI sites located in the intron are used for miRNA expression. Transgenes are cloned between the unique AgeI and XbaI sites. Additionally, both vectors contain elements necessary for the HIV-1 life cycle including the long-terminal repeats (LTRs), ψ packaging signal, Rev response element (RRE) and the central polypurine tract (cPPT) as well as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which enhances vector expression. The 3′ LTR bears a large deletion in U3 rendering the lentivirus self-inactivating (SIN).
    Ptripz Xrn1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ptripz xrn1 - by Bioz Stars, 2020-05
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    85
    Thermo Fisher shciap1 ptripz
    Schematic representation of control forward <t>(pTRIPZ)</t> and novel reverse orientation (pTREX) lentiviral expression vectors. (A) The forward orientation lentiviral vector pTRIPZ is commercially available (GE Healthcare/Dharmacon). pTRIPZ allows expression of a transgene cloned between unique AgeI and ClaI sites and miRNAs cloned between unique XhoI and EcoRI sites present in the transgene 3′UTR. Both are transcribed from a <t>tet-inducible</t> minimal CMV promoter (TetO/CMV). A 3′ Ubiquitin C promoter (UBC) drives the constitutive expression of the reverse tetracycline transactivator 3 (rtTA3) protein as well as a puromycin (Puro) selectable marker located 3′ to an internal ribosome entry site (IRES). (B) The pTREX lentiviral expression vector was generated from pTRIPZ by inverting sequences encompassing the TetO/CMV and inserting an intron derived from the rat preproinsulin II gene 5′ of the inserted transgene. pTREX also contains a poly(A) addition site located 3′ to the inserted transgene. Unique XhoI and EcoRI sites located in the intron are used for miRNA expression. Transgenes are cloned between the unique AgeI and XbaI sites. Additionally, both vectors contain elements necessary for the HIV-1 life cycle including the long-terminal repeats (LTRs), ψ packaging signal, Rev response element (RRE) and the central polypurine tract (cPPT) as well as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which enhances vector expression. The 3′ LTR bears a large deletion in U3 rendering the lentivirus self-inactivating (SIN).
    Shciap1 Ptripz, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    shciap1 ptripz - by Bioz Stars, 2020-05
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    88
    Thermo Fisher ptripz shp21
    Schematic representation of control forward <t>(pTRIPZ)</t> and novel reverse orientation (pTREX) lentiviral expression vectors. (A) The forward orientation lentiviral vector pTRIPZ is commercially available (GE Healthcare/Dharmacon). pTRIPZ allows expression of a transgene cloned between unique AgeI and ClaI sites and miRNAs cloned between unique XhoI and EcoRI sites present in the transgene 3′UTR. Both are transcribed from a <t>tet-inducible</t> minimal CMV promoter (TetO/CMV). A 3′ Ubiquitin C promoter (UBC) drives the constitutive expression of the reverse tetracycline transactivator 3 (rtTA3) protein as well as a puromycin (Puro) selectable marker located 3′ to an internal ribosome entry site (IRES). (B) The pTREX lentiviral expression vector was generated from pTRIPZ by inverting sequences encompassing the TetO/CMV and inserting an intron derived from the rat preproinsulin II gene 5′ of the inserted transgene. pTREX also contains a poly(A) addition site located 3′ to the inserted transgene. Unique XhoI and EcoRI sites located in the intron are used for miRNA expression. Transgenes are cloned between the unique AgeI and XbaI sites. Additionally, both vectors contain elements necessary for the HIV-1 life cycle including the long-terminal repeats (LTRs), ψ packaging signal, Rev response element (RRE) and the central polypurine tract (cPPT) as well as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which enhances vector expression. The 3′ LTR bears a large deletion in U3 rendering the lentivirus self-inactivating (SIN).
    Ptripz Shp21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ptripz shp21 - by Bioz Stars, 2020-05
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    90
    Thermo Fisher ptripz shxrn1
    Schematic representation of control forward <t>(pTRIPZ)</t> and novel reverse orientation (pTREX) lentiviral expression vectors. (A) The forward orientation lentiviral vector pTRIPZ is commercially available (GE Healthcare/Dharmacon). pTRIPZ allows expression of a transgene cloned between unique AgeI and ClaI sites and miRNAs cloned between unique XhoI and EcoRI sites present in the transgene 3′UTR. Both are transcribed from a <t>tet-inducible</t> minimal CMV promoter (TetO/CMV). A 3′ Ubiquitin C promoter (UBC) drives the constitutive expression of the reverse tetracycline transactivator 3 (rtTA3) protein as well as a puromycin (Puro) selectable marker located 3′ to an internal ribosome entry site (IRES). (B) The pTREX lentiviral expression vector was generated from pTRIPZ by inverting sequences encompassing the TetO/CMV and inserting an intron derived from the rat preproinsulin II gene 5′ of the inserted transgene. pTREX also contains a poly(A) addition site located 3′ to the inserted transgene. Unique XhoI and EcoRI sites located in the intron are used for miRNA expression. Transgenes are cloned between the unique AgeI and XbaI sites. Additionally, both vectors contain elements necessary for the HIV-1 life cycle including the long-terminal repeats (LTRs), ψ packaging signal, Rev response element (RRE) and the central polypurine tract (cPPT) as well as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which enhances vector expression. The 3′ LTR bears a large deletion in U3 rendering the lentivirus self-inactivating (SIN).
    Ptripz Shxrn1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    GE Healthcare ptripz empty
    Schematic representation of control forward <t>(pTRIPZ)</t> and novel reverse orientation (pTREX) lentiviral expression vectors. (A) The forward orientation lentiviral vector pTRIPZ is commercially available (GE Healthcare/Dharmacon). pTRIPZ allows expression of a transgene cloned between unique AgeI and ClaI sites and miRNAs cloned between unique XhoI and EcoRI sites present in the transgene 3′UTR. Both are transcribed from a <t>tet-inducible</t> minimal CMV promoter (TetO/CMV). A 3′ Ubiquitin C promoter (UBC) drives the constitutive expression of the reverse tetracycline transactivator 3 (rtTA3) protein as well as a puromycin (Puro) selectable marker located 3′ to an internal ribosome entry site (IRES). (B) The pTREX lentiviral expression vector was generated from pTRIPZ by inverting sequences encompassing the TetO/CMV and inserting an intron derived from the rat preproinsulin II gene 5′ of the inserted transgene. pTREX also contains a poly(A) addition site located 3′ to the inserted transgene. Unique XhoI and EcoRI sites located in the intron are used for miRNA expression. Transgenes are cloned between the unique AgeI and XbaI sites. Additionally, both vectors contain elements necessary for the HIV-1 life cycle including the long-terminal repeats (LTRs), ψ packaging signal, Rev response element (RRE) and the central polypurine tract (cPPT) as well as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which enhances vector expression. The 3′ LTR bears a large deletion in U3 rendering the lentivirus self-inactivating (SIN).
    Ptripz Empty, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher ptripz shrna against phgdh
    Schematic representation of control forward <t>(pTRIPZ)</t> and novel reverse orientation (pTREX) lentiviral expression vectors. (A) The forward orientation lentiviral vector pTRIPZ is commercially available (GE Healthcare/Dharmacon). pTRIPZ allows expression of a transgene cloned between unique AgeI and ClaI sites and miRNAs cloned between unique XhoI and EcoRI sites present in the transgene 3′UTR. Both are transcribed from a <t>tet-inducible</t> minimal CMV promoter (TetO/CMV). A 3′ Ubiquitin C promoter (UBC) drives the constitutive expression of the reverse tetracycline transactivator 3 (rtTA3) protein as well as a puromycin (Puro) selectable marker located 3′ to an internal ribosome entry site (IRES). (B) The pTREX lentiviral expression vector was generated from pTRIPZ by inverting sequences encompassing the TetO/CMV and inserting an intron derived from the rat preproinsulin II gene 5′ of the inserted transgene. pTREX also contains a poly(A) addition site located 3′ to the inserted transgene. Unique XhoI and EcoRI sites located in the intron are used for miRNA expression. Transgenes are cloned between the unique AgeI and XbaI sites. Additionally, both vectors contain elements necessary for the HIV-1 life cycle including the long-terminal repeats (LTRs), ψ packaging signal, Rev response element (RRE) and the central polypurine tract (cPPT) as well as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which enhances vector expression. The 3′ LTR bears a large deletion in U3 rendering the lentivirus self-inactivating (SIN).
    Ptripz Shrna Against Phgdh, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher ptripz vector
    Dose-dependent effect of E2F1 on DNA synthesis and content. Cells expressing <t>pTRIPZ-YFP-ER-E2F1</t> were starved of growth factors for 2 days and then treated with a combination of 1 μ g/ml doxycycline, 90 nM OHT or control starvation medium in the presence of 10 μ M EdU. Cells were fixed and stained for DNA content (DAPI) and DNA synthesis (EdU incorporation) 20 h later. ( a ) YFP/EdU distribution from induced HME cells. Red horizontal dashed line represents the boundary between EdU-positive and EdU-negative cells. Regions of low, intermediate and high YFP used for plotting DNA/EdU distributions in ( c ) are marked with red boxes. ( b ) YFP/DNA distribution from induced HME cells. Red horizontal line represents the boundary between cells with 2 N and > 2 N DNA content. ( c ) DNA/EdU distributions from 25 000 cells with either low, intermediate or high YFP marked with red boxes in a . The percentage of EdU-positive cells is indicated. ( d ) Data in a were analyzed by bin analysis where cells were divided into 21 bins according to their YFP fluorescence. The first bin contained all cells with YFP fluorescence under 100 a.u. (range of uninduced samples). Red dots represent proportion of EdU-positive cells for each YFP bin. Blue dot represents uninduced control. ( e ) Bin analysis as in d for DNA content. ( f ) Analysis as in d for three cancer cell lines and HME normal cells using 60 YFP bins. ( g ) Analysis as in e for three cancer cell lines and HME normal cells using 60 YFP bins
    Ptripz Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 566 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher ptripz lentiviral system
    Dose-dependent effect of E2F1 on DNA synthesis and content. Cells expressing <t>pTRIPZ-YFP-ER-E2F1</t> were starved of growth factors for 2 days and then treated with a combination of 1 μ g/ml doxycycline, 90 nM OHT or control starvation medium in the presence of 10 μ M EdU. Cells were fixed and stained for DNA content (DAPI) and DNA synthesis (EdU incorporation) 20 h later. ( a ) YFP/EdU distribution from induced HME cells. Red horizontal dashed line represents the boundary between EdU-positive and EdU-negative cells. Regions of low, intermediate and high YFP used for plotting DNA/EdU distributions in ( c ) are marked with red boxes. ( b ) YFP/DNA distribution from induced HME cells. Red horizontal line represents the boundary between cells with 2 N and > 2 N DNA content. ( c ) DNA/EdU distributions from 25 000 cells with either low, intermediate or high YFP marked with red boxes in a . The percentage of EdU-positive cells is indicated. ( d ) Data in a were analyzed by bin analysis where cells were divided into 21 bins according to their YFP fluorescence. The first bin contained all cells with YFP fluorescence under 100 a.u. (range of uninduced samples). Red dots represent proportion of EdU-positive cells for each YFP bin. Blue dot represents uninduced control. ( e ) Bin analysis as in d for DNA content. ( f ) Analysis as in d for three cancer cell lines and HME normal cells using 60 YFP bins. ( g ) Analysis as in e for three cancer cell lines and HME normal cells using 60 YFP bins
    Ptripz Lentiviral System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    TaKaRa ptripz vector
    Dose-dependent effect of E2F1 on DNA synthesis and content. Cells expressing <t>pTRIPZ-YFP-ER-E2F1</t> were starved of growth factors for 2 days and then treated with a combination of 1 μ g/ml doxycycline, 90 nM OHT or control starvation medium in the presence of 10 μ M EdU. Cells were fixed and stained for DNA content (DAPI) and DNA synthesis (EdU incorporation) 20 h later. ( a ) YFP/EdU distribution from induced HME cells. Red horizontal dashed line represents the boundary between EdU-positive and EdU-negative cells. Regions of low, intermediate and high YFP used for plotting DNA/EdU distributions in ( c ) are marked with red boxes. ( b ) YFP/DNA distribution from induced HME cells. Red horizontal line represents the boundary between cells with 2 N and > 2 N DNA content. ( c ) DNA/EdU distributions from 25 000 cells with either low, intermediate or high YFP marked with red boxes in a . The percentage of EdU-positive cells is indicated. ( d ) Data in a were analyzed by bin analysis where cells were divided into 21 bins according to their YFP fluorescence. The first bin contained all cells with YFP fluorescence under 100 a.u. (range of uninduced samples). Red dots represent proportion of EdU-positive cells for each YFP bin. Blue dot represents uninduced control. ( e ) Bin analysis as in d for DNA content. ( f ) Analysis as in d for three cancer cell lines and HME normal cells using 60 YFP bins. ( g ) Analysis as in e for three cancer cell lines and HME normal cells using 60 YFP bins
    Ptripz Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher ptripz shrna
    Dose-dependent effect of E2F1 on DNA synthesis and content. Cells expressing <t>pTRIPZ-YFP-ER-E2F1</t> were starved of growth factors for 2 days and then treated with a combination of 1 μ g/ml doxycycline, 90 nM OHT or control starvation medium in the presence of 10 μ M EdU. Cells were fixed and stained for DNA content (DAPI) and DNA synthesis (EdU incorporation) 20 h later. ( a ) YFP/EdU distribution from induced HME cells. Red horizontal dashed line represents the boundary between EdU-positive and EdU-negative cells. Regions of low, intermediate and high YFP used for plotting DNA/EdU distributions in ( c ) are marked with red boxes. ( b ) YFP/DNA distribution from induced HME cells. Red horizontal line represents the boundary between cells with 2 N and > 2 N DNA content. ( c ) DNA/EdU distributions from 25 000 cells with either low, intermediate or high YFP marked with red boxes in a . The percentage of EdU-positive cells is indicated. ( d ) Data in a were analyzed by bin analysis where cells were divided into 21 bins according to their YFP fluorescence. The first bin contained all cells with YFP fluorescence under 100 a.u. (range of uninduced samples). Red dots represent proportion of EdU-positive cells for each YFP bin. Blue dot represents uninduced control. ( e ) Bin analysis as in d for DNA content. ( f ) Analysis as in d for three cancer cell lines and HME normal cells using 60 YFP bins. ( g ) Analysis as in e for three cancer cell lines and HME normal cells using 60 YFP bins
    Ptripz Shrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher ptripz sh2 h2afj
    The H2A.J C-terminal sequence is required for its function. WI-38 hTERT cells were infected with pGIPz lentiviruses expressing a No Target shRNA (sh-NT) or an sh3- <t>H2AFJ</t> RNA. The sh3- H2AFJ cell line was super-infected with <t>pTRIPz</t> lentiviruses expressing sh3-resistant H2AFJ cDNAs encoding WT-H2A.J or a mutant H2A.J in which the C-terminus of H2A.J was substituted with the C terminus of canonical H2A type 1 (H2A.J-Cter-H2A). Both the WT and mutant H2A.J contained an N-terminal Flag-HA tag. RNA was isolated from cells induced into senescence by etoposide treatment for 3 weeks and compared to levels in proliferating sh-No Target cells. ( a ) Schematic of V11 and C-terminal aa sequences of H2A.J and H2A.J-C-ter-H2A. ( b ) RT–qPCR quantification of inflammatory gene expression for three biological replicates (independent cultures at different dates) for the indicated genes. The induction ratio for these genes in senescent versus proliferating sh-NT cells varied between biological replicates. The extent to which ectopic WT-H2A.J restored defective gene expression in the sh- H2AFJ knockdown cells also varied between biological replicates. However, the H2A.J-Cter-H2A mutant was ineffective at restoring inflammatory gene expression, indicating the functional importance of the specific H2A.J C-terminal sequence. For each biological replicate, the mean and s.d. are shown for three qPCR technical replicates.
    Ptripz Sh2 H2afj, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GE Healthcare ptripz vector
    The H2A.J C-terminal sequence is required for its function. WI-38 hTERT cells were infected with pGIPz lentiviruses expressing a No Target shRNA (sh-NT) or an sh3- <t>H2AFJ</t> RNA. The sh3- H2AFJ cell line was super-infected with <t>pTRIPz</t> lentiviruses expressing sh3-resistant H2AFJ cDNAs encoding WT-H2A.J or a mutant H2A.J in which the C-terminus of H2A.J was substituted with the C terminus of canonical H2A type 1 (H2A.J-Cter-H2A). Both the WT and mutant H2A.J contained an N-terminal Flag-HA tag. RNA was isolated from cells induced into senescence by etoposide treatment for 3 weeks and compared to levels in proliferating sh-No Target cells. ( a ) Schematic of V11 and C-terminal aa sequences of H2A.J and H2A.J-C-ter-H2A. ( b ) RT–qPCR quantification of inflammatory gene expression for three biological replicates (independent cultures at different dates) for the indicated genes. The induction ratio for these genes in senescent versus proliferating sh-NT cells varied between biological replicates. The extent to which ectopic WT-H2A.J restored defective gene expression in the sh- H2AFJ knockdown cells also varied between biological replicates. However, the H2A.J-Cter-H2A mutant was ineffective at restoring inflammatory gene expression, indicating the functional importance of the specific H2A.J C-terminal sequence. For each biological replicate, the mean and s.d. are shown for three qPCR technical replicates.
    Ptripz Vector, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 16 article reviews
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    ptripz vector - by Bioz Stars, 2020-05
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    90
    Horizon Discovery ptripz vector
    The H2A.J C-terminal sequence is required for its function. WI-38 hTERT cells were infected with pGIPz lentiviruses expressing a No Target shRNA (sh-NT) or an sh3- <t>H2AFJ</t> RNA. The sh3- H2AFJ cell line was super-infected with <t>pTRIPz</t> lentiviruses expressing sh3-resistant H2AFJ cDNAs encoding WT-H2A.J or a mutant H2A.J in which the C-terminus of H2A.J was substituted with the C terminus of canonical H2A type 1 (H2A.J-Cter-H2A). Both the WT and mutant H2A.J contained an N-terminal Flag-HA tag. RNA was isolated from cells induced into senescence by etoposide treatment for 3 weeks and compared to levels in proliferating sh-No Target cells. ( a ) Schematic of V11 and C-terminal aa sequences of H2A.J and H2A.J-C-ter-H2A. ( b ) RT–qPCR quantification of inflammatory gene expression for three biological replicates (independent cultures at different dates) for the indicated genes. The induction ratio for these genes in senescent versus proliferating sh-NT cells varied between biological replicates. The extent to which ectopic WT-H2A.J restored defective gene expression in the sh- H2AFJ knockdown cells also varied between biological replicates. However, the H2A.J-Cter-H2A mutant was ineffective at restoring inflammatory gene expression, indicating the functional importance of the specific H2A.J C-terminal sequence. For each biological replicate, the mean and s.d. are shown for three qPCR technical replicates.
    Ptripz Vector, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 90/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 82 article reviews
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    ptripz vector - by Bioz Stars, 2020-05
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    91
    Thermo Fisher ptripz sh3 h2afj
    H2A.J is required for normal expression of SASP genes. ( a ) GSEA enrichment plot showing the ranking of 38 SASP genes within the set of genes differentially-expressed on knock-down of H2A.J in senescent WI-38hTERT fibroblasts. ( b ) Corresponding heat maps for the 38 SASP genes. Each row represents an independent biological replicate of cells expressing <t>sh3-</t> <t>H2AFJ</t> or sh-NoTarget. Genes are listed by highest (left) to lowest (right) expression in the sh-NoTarget cells. ( c ) RT–qPCR verification of the effect of H2A.J knock-down on CXCL1 , CXCL2 , CXCL3 , and IL1B expression in senescent cells (SEN). Values were normalized to proliferating fibroblasts expressing an sh-No Target RNA (PRO-NT). Shown are 3 biological replicates for senescent cells expressing No Target or sh- H2AFJ RNAs. Error bars represent mean±s.d. t -tests with * P
    Ptripz Sh3 H2afj, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ptripz sh3 h2afj/product/Thermo Fisher
    Average 91 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Intracranial (i.c.) xenograft formation assay of U251-NS Kaplan-Meier survival curves were was plotted from overall survivals of mice following i.c. implantation of U251-NS infectants of pTRIPZ-lentiviral constructs of vector, wild-type and variants of EFEMP1.

    Journal: Oncoscience

    Article Title: Weaponizing human EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) for 21st century cancer therapeutics

    doi: 10.18632/oncoscience.306

    Figure Lengend Snippet: Intracranial (i.c.) xenograft formation assay of U251-NS Kaplan-Meier survival curves were was plotted from overall survivals of mice following i.c. implantation of U251-NS infectants of pTRIPZ-lentiviral constructs of vector, wild-type and variants of EFEMP1.

    Article Snippet: A shuttle vector had been constructed so as to introduce an internal ribosome entry site for expression of EFEMP1 wildtype and variant proteins in pTRIPZ (Open Biosystems, Huntsville, AL) under the same promoter used for red fluorescent protein (RFP).

    Techniques: Tube Formation Assay, Mouse Assay, Construct, Plasmid Preparation

    Subcutaneous (s.c.) xenograft formation assay of U251 A. comparison of s.c. tumor weights of U251 stable transfectants of wild-type EFEMP1 and EFEMP1 variants. P, parental un-transfected U251. B. comparison of s.c. tumor weights of U251 lentiviral infectants of pTRIPZ-lentiviral constructs of vector, wild-type and variants of EFEMP1.

    Journal: Oncoscience

    Article Title: Weaponizing human EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) for 21st century cancer therapeutics

    doi: 10.18632/oncoscience.306

    Figure Lengend Snippet: Subcutaneous (s.c.) xenograft formation assay of U251 A. comparison of s.c. tumor weights of U251 stable transfectants of wild-type EFEMP1 and EFEMP1 variants. P, parental un-transfected U251. B. comparison of s.c. tumor weights of U251 lentiviral infectants of pTRIPZ-lentiviral constructs of vector, wild-type and variants of EFEMP1.

    Article Snippet: A shuttle vector had been constructed so as to introduce an internal ribosome entry site for expression of EFEMP1 wildtype and variant proteins in pTRIPZ (Open Biosystems, Huntsville, AL) under the same promoter used for red fluorescent protein (RFP).

    Techniques: Tube Formation Assay, Transfection, Construct, Plasmid Preparation

    Immunoblotting EGFR and NOTCH 1 Whole cell lysate of U251infectants of pTRIPZ-lentiviral constructs of wild-type and variants of EFEMP1 following a 3-day cultures in medium with or without addition of doxycycline (Dox) were blotted and detected by antibodies of EGFR and NOTCH1. ACTB was detected to control equal protein loading. Since the transduced cells were kept in the “off” condition by omitting doxycycline, the un-induced cells served as the negative control for induced cells. In addition, construct E10, the 1-bp altered variant of EFEMP1 that showed minimal functional alteration of EFEMP1, was included as a negative control.

    Journal: Oncoscience

    Article Title: Weaponizing human EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) for 21st century cancer therapeutics

    doi: 10.18632/oncoscience.306

    Figure Lengend Snippet: Immunoblotting EGFR and NOTCH 1 Whole cell lysate of U251infectants of pTRIPZ-lentiviral constructs of wild-type and variants of EFEMP1 following a 3-day cultures in medium with or without addition of doxycycline (Dox) were blotted and detected by antibodies of EGFR and NOTCH1. ACTB was detected to control equal protein loading. Since the transduced cells were kept in the “off” condition by omitting doxycycline, the un-induced cells served as the negative control for induced cells. In addition, construct E10, the 1-bp altered variant of EFEMP1 that showed minimal functional alteration of EFEMP1, was included as a negative control.

    Article Snippet: A shuttle vector had been constructed so as to introduce an internal ribosome entry site for expression of EFEMP1 wildtype and variant proteins in pTRIPZ (Open Biosystems, Huntsville, AL) under the same promoter used for red fluorescent protein (RFP).

    Techniques: Construct, Negative Control, Variant Assay, Functional Assay

    Schematic representation of control forward (pTRIPZ) and novel reverse orientation (pTREX) lentiviral expression vectors. (A) The forward orientation lentiviral vector pTRIPZ is commercially available (GE Healthcare/Dharmacon). pTRIPZ allows expression of a transgene cloned between unique AgeI and ClaI sites and miRNAs cloned between unique XhoI and EcoRI sites present in the transgene 3′UTR. Both are transcribed from a tet-inducible minimal CMV promoter (TetO/CMV). A 3′ Ubiquitin C promoter (UBC) drives the constitutive expression of the reverse tetracycline transactivator 3 (rtTA3) protein as well as a puromycin (Puro) selectable marker located 3′ to an internal ribosome entry site (IRES). (B) The pTREX lentiviral expression vector was generated from pTRIPZ by inverting sequences encompassing the TetO/CMV and inserting an intron derived from the rat preproinsulin II gene 5′ of the inserted transgene. pTREX also contains a poly(A) addition site located 3′ to the inserted transgene. Unique XhoI and EcoRI sites located in the intron are used for miRNA expression. Transgenes are cloned between the unique AgeI and XbaI sites. Additionally, both vectors contain elements necessary for the HIV-1 life cycle including the long-terminal repeats (LTRs), ψ packaging signal, Rev response element (RRE) and the central polypurine tract (cPPT) as well as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which enhances vector expression. The 3′ LTR bears a large deletion in U3 rendering the lentivirus self-inactivating (SIN).

    Journal: RNA Biology

    Article Title: A lentiviral vector bearing a reverse intron demonstrates superior expression of both proteins and microRNAs

    doi: 10.1080/15476286.2017.1334755

    Figure Lengend Snippet: Schematic representation of control forward (pTRIPZ) and novel reverse orientation (pTREX) lentiviral expression vectors. (A) The forward orientation lentiviral vector pTRIPZ is commercially available (GE Healthcare/Dharmacon). pTRIPZ allows expression of a transgene cloned between unique AgeI and ClaI sites and miRNAs cloned between unique XhoI and EcoRI sites present in the transgene 3′UTR. Both are transcribed from a tet-inducible minimal CMV promoter (TetO/CMV). A 3′ Ubiquitin C promoter (UBC) drives the constitutive expression of the reverse tetracycline transactivator 3 (rtTA3) protein as well as a puromycin (Puro) selectable marker located 3′ to an internal ribosome entry site (IRES). (B) The pTREX lentiviral expression vector was generated from pTRIPZ by inverting sequences encompassing the TetO/CMV and inserting an intron derived from the rat preproinsulin II gene 5′ of the inserted transgene. pTREX also contains a poly(A) addition site located 3′ to the inserted transgene. Unique XhoI and EcoRI sites located in the intron are used for miRNA expression. Transgenes are cloned between the unique AgeI and XbaI sites. Additionally, both vectors contain elements necessary for the HIV-1 life cycle including the long-terminal repeats (LTRs), ψ packaging signal, Rev response element (RRE) and the central polypurine tract (cPPT) as well as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which enhances vector expression. The 3′ LTR bears a large deletion in U3 rendering the lentivirus self-inactivating (SIN).

    Article Snippet: pTREX was created by PCR amplification of the Tet-inducible minimal TetO/CMV promoter and transgene TurboRFP from pTRIPZ (RHS4696, Dharmacon) followed by cloning in the reverse orientation at the same location in pTRIPZ.

    Techniques: Expressing, Plasmid Preparation, Clone Assay, Marker, Generated, Derivative Assay

    pTREX expresses a higher level of a marker gene when pri-miRNAs are present in cis . pTREX and pTRIPZ vectors bearing a Thy1.1 transgene were packaged in NoDice/ΔPKR cells and transduced into HeLa cells. Transduced cells were selected for puromycin resistance, and then treated with Dox for 3 d. Thy1.1 positivity was measured with an APC-conjugated antibody by flow cytometry. Thy1.1 histograms are shown for (A) empty vector, (B) miR-155, (C) miR-BHRF1–123, and (D) miR-K1–5-transduced HeLa cells. pTREX is shown in blue and pTRIPZ is shown in red. The average of 3 independent experiments is shown as (E) percent Thy1.1+ cells and (F) MFI of positive cells. Student's t-test, *** p

    Journal: RNA Biology

    Article Title: A lentiviral vector bearing a reverse intron demonstrates superior expression of both proteins and microRNAs

    doi: 10.1080/15476286.2017.1334755

    Figure Lengend Snippet: pTREX expresses a higher level of a marker gene when pri-miRNAs are present in cis . pTREX and pTRIPZ vectors bearing a Thy1.1 transgene were packaged in NoDice/ΔPKR cells and transduced into HeLa cells. Transduced cells were selected for puromycin resistance, and then treated with Dox for 3 d. Thy1.1 positivity was measured with an APC-conjugated antibody by flow cytometry. Thy1.1 histograms are shown for (A) empty vector, (B) miR-155, (C) miR-BHRF1–123, and (D) miR-K1–5-transduced HeLa cells. pTREX is shown in blue and pTRIPZ is shown in red. The average of 3 independent experiments is shown as (E) percent Thy1.1+ cells and (F) MFI of positive cells. Student's t-test, *** p

    Article Snippet: pTREX was created by PCR amplification of the Tet-inducible minimal TetO/CMV promoter and transgene TurboRFP from pTRIPZ (RHS4696, Dharmacon) followed by cloning in the reverse orientation at the same location in pTRIPZ.

    Techniques: Marker, Flow Cytometry, Cytometry, Plasmid Preparation

    pTREX shows superior transgene expression when compared with pTRIPZ. (A-D) 293T cells were transduced with the lentiviral vectors pTRIPZ or pTREX expressing GFP as a transgene and selected with puromycin for 1 week. GFP expression was then induced by treatment with Dox for 3 d or the cells were maintained in the absence of Dox. GFP expression was then analyzed by flow cytometry. GFP signal histograms are shown for (A) pTRIPZ-transduced cells, and (B) pTREX-transduced cells, with counts of Dox-induced cells in green, and no Dox controls in gray. The average of 4 independent transductions is shown as (C) mean fluorescence intensity (MFI) of GFP+ cells or (D) percent GFP+ of the whole population. Data were analyzed by a Student's t-test; ** p

    Journal: RNA Biology

    Article Title: A lentiviral vector bearing a reverse intron demonstrates superior expression of both proteins and microRNAs

    doi: 10.1080/15476286.2017.1334755

    Figure Lengend Snippet: pTREX shows superior transgene expression when compared with pTRIPZ. (A-D) 293T cells were transduced with the lentiviral vectors pTRIPZ or pTREX expressing GFP as a transgene and selected with puromycin for 1 week. GFP expression was then induced by treatment with Dox for 3 d or the cells were maintained in the absence of Dox. GFP expression was then analyzed by flow cytometry. GFP signal histograms are shown for (A) pTRIPZ-transduced cells, and (B) pTREX-transduced cells, with counts of Dox-induced cells in green, and no Dox controls in gray. The average of 4 independent transductions is shown as (C) mean fluorescence intensity (MFI) of GFP+ cells or (D) percent GFP+ of the whole population. Data were analyzed by a Student's t-test; ** p

    Article Snippet: pTREX was created by PCR amplification of the Tet-inducible minimal TetO/CMV promoter and transgene TurboRFP from pTRIPZ (RHS4696, Dharmacon) followed by cloning in the reverse orientation at the same location in pTRIPZ.

    Techniques: Expressing, Transduction, Flow Cytometry, Cytometry, Fluorescence

    Dose-dependent effect of E2F1 on DNA synthesis and content. Cells expressing pTRIPZ-YFP-ER-E2F1 were starved of growth factors for 2 days and then treated with a combination of 1 μ g/ml doxycycline, 90 nM OHT or control starvation medium in the presence of 10 μ M EdU. Cells were fixed and stained for DNA content (DAPI) and DNA synthesis (EdU incorporation) 20 h later. ( a ) YFP/EdU distribution from induced HME cells. Red horizontal dashed line represents the boundary between EdU-positive and EdU-negative cells. Regions of low, intermediate and high YFP used for plotting DNA/EdU distributions in ( c ) are marked with red boxes. ( b ) YFP/DNA distribution from induced HME cells. Red horizontal line represents the boundary between cells with 2 N and > 2 N DNA content. ( c ) DNA/EdU distributions from 25 000 cells with either low, intermediate or high YFP marked with red boxes in a . The percentage of EdU-positive cells is indicated. ( d ) Data in a were analyzed by bin analysis where cells were divided into 21 bins according to their YFP fluorescence. The first bin contained all cells with YFP fluorescence under 100 a.u. (range of uninduced samples). Red dots represent proportion of EdU-positive cells for each YFP bin. Blue dot represents uninduced control. ( e ) Bin analysis as in d for DNA content. ( f ) Analysis as in d for three cancer cell lines and HME normal cells using 60 YFP bins. ( g ) Analysis as in e for three cancer cell lines and HME normal cells using 60 YFP bins

    Journal: Cell Death and Differentiation

    Article Title: Expression level is a key determinant of E2F1-mediated cell fate

    doi: 10.1038/cdd.2017.12

    Figure Lengend Snippet: Dose-dependent effect of E2F1 on DNA synthesis and content. Cells expressing pTRIPZ-YFP-ER-E2F1 were starved of growth factors for 2 days and then treated with a combination of 1 μ g/ml doxycycline, 90 nM OHT or control starvation medium in the presence of 10 μ M EdU. Cells were fixed and stained for DNA content (DAPI) and DNA synthesis (EdU incorporation) 20 h later. ( a ) YFP/EdU distribution from induced HME cells. Red horizontal dashed line represents the boundary between EdU-positive and EdU-negative cells. Regions of low, intermediate and high YFP used for plotting DNA/EdU distributions in ( c ) are marked with red boxes. ( b ) YFP/DNA distribution from induced HME cells. Red horizontal line represents the boundary between cells with 2 N and > 2 N DNA content. ( c ) DNA/EdU distributions from 25 000 cells with either low, intermediate or high YFP marked with red boxes in a . The percentage of EdU-positive cells is indicated. ( d ) Data in a were analyzed by bin analysis where cells were divided into 21 bins according to their YFP fluorescence. The first bin contained all cells with YFP fluorescence under 100 a.u. (range of uninduced samples). Red dots represent proportion of EdU-positive cells for each YFP bin. Blue dot represents uninduced control. ( e ) Bin analysis as in d for DNA content. ( f ) Analysis as in d for three cancer cell lines and HME normal cells using 60 YFP bins. ( g ) Analysis as in e for three cancer cell lines and HME normal cells using 60 YFP bins

    Article Snippet: pEYFP-ER-E2F1 construct was generated by inserting the Nhe I digestion fragment containing the ER-E2F1 cassette from pBabePuro-HA.ER.E2F1 (a gift from Dr. Kristian Helin) into the compatible Xba I digestion site of the pEYFP-C1 vector (Clontech, Mountain View, CA, USA). pTRIPZ-YFP-ER-E2F1 construct was generated by inserting the YFP-ER-E2F1 cassette from pEYFP-ER-E2F1 into the pTRIPZ vector (Open Biosystems, Lafayette, CO, USA) between the Age I and Mlu I sites replacing the TurboRFP and shRNA cassette.

    Techniques: DNA Synthesis, Expressing, Staining, Fluorescence

    E2F1 induces mitotic arrest in multiple cell lines. ( a ) HME cells expressing pTRIPZ-YFP-ER-E2F1 in full growth factor containing medium were treated with a combination of 1 μ g/ml doxycycline and 90 nM OHT for 24 h. Cells were stained with anti-phospho-histone H3 antibody (mitotic marker) and analyzed by flow cytometry. ( b ) Six cell lines expressing pTRIPZ-YFP-ER-E2F1 in full serum-containing medium were treated with a combination of 1 μ for the raw data

    Journal: Cell Death and Differentiation

    Article Title: Expression level is a key determinant of E2F1-mediated cell fate

    doi: 10.1038/cdd.2017.12

    Figure Lengend Snippet: E2F1 induces mitotic arrest in multiple cell lines. ( a ) HME cells expressing pTRIPZ-YFP-ER-E2F1 in full growth factor containing medium were treated with a combination of 1 μ g/ml doxycycline and 90 nM OHT for 24 h. Cells were stained with anti-phospho-histone H3 antibody (mitotic marker) and analyzed by flow cytometry. ( b ) Six cell lines expressing pTRIPZ-YFP-ER-E2F1 in full serum-containing medium were treated with a combination of 1 μ for the raw data

    Article Snippet: pEYFP-ER-E2F1 construct was generated by inserting the Nhe I digestion fragment containing the ER-E2F1 cassette from pBabePuro-HA.ER.E2F1 (a gift from Dr. Kristian Helin) into the compatible Xba I digestion site of the pEYFP-C1 vector (Clontech, Mountain View, CA, USA). pTRIPZ-YFP-ER-E2F1 construct was generated by inserting the YFP-ER-E2F1 cassette from pEYFP-ER-E2F1 into the pTRIPZ vector (Open Biosystems, Lafayette, CO, USA) between the Age I and Mlu I sites replacing the TurboRFP and shRNA cassette.

    Techniques: Expressing, Staining, Marker, Flow Cytometry, Cytometry

    Experimental system for studies of E2F1-mediated cell fates at a single-cell resolution. ( a ) Schematic of YFP–ER–E2F1 fusion protein. ( b ) U2OS cells stably expressing pEYFP-ER-E2F1 were serum starved for 24 h and then treated with 2 μ M tamoxifen in starvation medium. Cells were collected at indicated time points and expression of BIM, APAF1, FOXO3 and CASP3 analyzed by real-time qPCR. ( c ) U2OS cells stably expressing pEYFP-ER-E2F1 were serum starved for 24 h and then treated with 2 μ M tamoxifen in starvation medium. Cells were collected at indicated time points and PARP cleavage was analyzed by western blot. ( d ) Images of U2OS cells expressing YFP-ER-E2F1 (green) and H2B-RFP (red) before (0 h), 4 h and 40 h after exposure to 3 μ M tamoxifen. ( e ) Schematic of pTRIPZ-YFP-ER-E2F1 construct. ( f and g ) U2OS pTRIPZ-YFP-ER-E2F1 cells were grown in full serum-containing growth medium and treated with 500 ng/ml doxycycline for 48 h followed by addition of 90 nM OHT for an additional 20 h. Cells from the indicated YFP fractions were sorted by flow cytometry and E2F1 expression was analyzed by quantitative RT-PCR. ( f ) Histogram of YFP expression and sorting gates in induced U2OS pTRIPZ-YFP-ER-E2F1 cells (red) and parental U2OS cells (blue). ( g ) Real-time quantitative RT-PCR analysis of total E2F1 (endogenous+YFP-ER-E2F1) in sorted fractions. Graph represents the relative E2F1 expression after normalization to GAPDH housekeeping control (mean and range of duplicate PCR reactions). IRES, internal ribosomal entry site; rtTA3, reverse tetracycline-transactivator 3; TRE, tetracycline-responsive element; UBC, ubiquitin C promoter

    Journal: Cell Death and Differentiation

    Article Title: Expression level is a key determinant of E2F1-mediated cell fate

    doi: 10.1038/cdd.2017.12

    Figure Lengend Snippet: Experimental system for studies of E2F1-mediated cell fates at a single-cell resolution. ( a ) Schematic of YFP–ER–E2F1 fusion protein. ( b ) U2OS cells stably expressing pEYFP-ER-E2F1 were serum starved for 24 h and then treated with 2 μ M tamoxifen in starvation medium. Cells were collected at indicated time points and expression of BIM, APAF1, FOXO3 and CASP3 analyzed by real-time qPCR. ( c ) U2OS cells stably expressing pEYFP-ER-E2F1 were serum starved for 24 h and then treated with 2 μ M tamoxifen in starvation medium. Cells were collected at indicated time points and PARP cleavage was analyzed by western blot. ( d ) Images of U2OS cells expressing YFP-ER-E2F1 (green) and H2B-RFP (red) before (0 h), 4 h and 40 h after exposure to 3 μ M tamoxifen. ( e ) Schematic of pTRIPZ-YFP-ER-E2F1 construct. ( f and g ) U2OS pTRIPZ-YFP-ER-E2F1 cells were grown in full serum-containing growth medium and treated with 500 ng/ml doxycycline for 48 h followed by addition of 90 nM OHT for an additional 20 h. Cells from the indicated YFP fractions were sorted by flow cytometry and E2F1 expression was analyzed by quantitative RT-PCR. ( f ) Histogram of YFP expression and sorting gates in induced U2OS pTRIPZ-YFP-ER-E2F1 cells (red) and parental U2OS cells (blue). ( g ) Real-time quantitative RT-PCR analysis of total E2F1 (endogenous+YFP-ER-E2F1) in sorted fractions. Graph represents the relative E2F1 expression after normalization to GAPDH housekeeping control (mean and range of duplicate PCR reactions). IRES, internal ribosomal entry site; rtTA3, reverse tetracycline-transactivator 3; TRE, tetracycline-responsive element; UBC, ubiquitin C promoter

    Article Snippet: pEYFP-ER-E2F1 construct was generated by inserting the Nhe I digestion fragment containing the ER-E2F1 cassette from pBabePuro-HA.ER.E2F1 (a gift from Dr. Kristian Helin) into the compatible Xba I digestion site of the pEYFP-C1 vector (Clontech, Mountain View, CA, USA). pTRIPZ-YFP-ER-E2F1 construct was generated by inserting the YFP-ER-E2F1 cassette from pEYFP-ER-E2F1 into the pTRIPZ vector (Open Biosystems, Lafayette, CO, USA) between the Age I and Mlu I sites replacing the TurboRFP and shRNA cassette.

    Techniques: Stable Transfection, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Construct, Flow Cytometry, Cytometry, Quantitative RT-PCR, Polymerase Chain Reaction

    The H2A.J C-terminal sequence is required for its function. WI-38 hTERT cells were infected with pGIPz lentiviruses expressing a No Target shRNA (sh-NT) or an sh3- H2AFJ RNA. The sh3- H2AFJ cell line was super-infected with pTRIPz lentiviruses expressing sh3-resistant H2AFJ cDNAs encoding WT-H2A.J or a mutant H2A.J in which the C-terminus of H2A.J was substituted with the C terminus of canonical H2A type 1 (H2A.J-Cter-H2A). Both the WT and mutant H2A.J contained an N-terminal Flag-HA tag. RNA was isolated from cells induced into senescence by etoposide treatment for 3 weeks and compared to levels in proliferating sh-No Target cells. ( a ) Schematic of V11 and C-terminal aa sequences of H2A.J and H2A.J-C-ter-H2A. ( b ) RT–qPCR quantification of inflammatory gene expression for three biological replicates (independent cultures at different dates) for the indicated genes. The induction ratio for these genes in senescent versus proliferating sh-NT cells varied between biological replicates. The extent to which ectopic WT-H2A.J restored defective gene expression in the sh- H2AFJ knockdown cells also varied between biological replicates. However, the H2A.J-Cter-H2A mutant was ineffective at restoring inflammatory gene expression, indicating the functional importance of the specific H2A.J C-terminal sequence. For each biological replicate, the mean and s.d. are shown for three qPCR technical replicates.

    Journal: Nature Communications

    Article Title: Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression

    doi: 10.1038/ncomms14995

    Figure Lengend Snippet: The H2A.J C-terminal sequence is required for its function. WI-38 hTERT cells were infected with pGIPz lentiviruses expressing a No Target shRNA (sh-NT) or an sh3- H2AFJ RNA. The sh3- H2AFJ cell line was super-infected with pTRIPz lentiviruses expressing sh3-resistant H2AFJ cDNAs encoding WT-H2A.J or a mutant H2A.J in which the C-terminus of H2A.J was substituted with the C terminus of canonical H2A type 1 (H2A.J-Cter-H2A). Both the WT and mutant H2A.J contained an N-terminal Flag-HA tag. RNA was isolated from cells induced into senescence by etoposide treatment for 3 weeks and compared to levels in proliferating sh-No Target cells. ( a ) Schematic of V11 and C-terminal aa sequences of H2A.J and H2A.J-C-ter-H2A. ( b ) RT–qPCR quantification of inflammatory gene expression for three biological replicates (independent cultures at different dates) for the indicated genes. The induction ratio for these genes in senescent versus proliferating sh-NT cells varied between biological replicates. The extent to which ectopic WT-H2A.J restored defective gene expression in the sh- H2AFJ knockdown cells also varied between biological replicates. However, the H2A.J-Cter-H2A mutant was ineffective at restoring inflammatory gene expression, indicating the functional importance of the specific H2A.J C-terminal sequence. For each biological replicate, the mean and s.d. are shown for three qPCR technical replicates.

    Article Snippet: shRNA knock-down of H2AFJ pTRIPz-sh-No Target (Thermo RHS4743), pTRIPz-sh2-H2AFJ (Thermo V3THS_302395) pTRIPz-sh3-H2AFJ (Thermo V3THS_351902), and pGIPz-sh-NoTarget (RHS4346) were purchased from Thermo Scientific.

    Techniques: Sequencing, Infection, Expressing, shRNA, Mutagenesis, Isolation, Quantitative RT-PCR, Functional Assay, Real-time Polymerase Chain Reaction

    H2A.J is required for normal expression of SASP genes. ( a ) GSEA enrichment plot showing the ranking of 38 SASP genes within the set of genes differentially-expressed on knock-down of H2A.J in senescent WI-38hTERT fibroblasts. ( b ) Corresponding heat maps for the 38 SASP genes. Each row represents an independent biological replicate of cells expressing sh3- H2AFJ or sh-NoTarget. Genes are listed by highest (left) to lowest (right) expression in the sh-NoTarget cells. ( c ) RT–qPCR verification of the effect of H2A.J knock-down on CXCL1 , CXCL2 , CXCL3 , and IL1B expression in senescent cells (SEN). Values were normalized to proliferating fibroblasts expressing an sh-No Target RNA (PRO-NT). Shown are 3 biological replicates for senescent cells expressing No Target or sh- H2AFJ RNAs. Error bars represent mean±s.d. t -tests with * P

    Journal: Nature Communications

    Article Title: Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression

    doi: 10.1038/ncomms14995

    Figure Lengend Snippet: H2A.J is required for normal expression of SASP genes. ( a ) GSEA enrichment plot showing the ranking of 38 SASP genes within the set of genes differentially-expressed on knock-down of H2A.J in senescent WI-38hTERT fibroblasts. ( b ) Corresponding heat maps for the 38 SASP genes. Each row represents an independent biological replicate of cells expressing sh3- H2AFJ or sh-NoTarget. Genes are listed by highest (left) to lowest (right) expression in the sh-NoTarget cells. ( c ) RT–qPCR verification of the effect of H2A.J knock-down on CXCL1 , CXCL2 , CXCL3 , and IL1B expression in senescent cells (SEN). Values were normalized to proliferating fibroblasts expressing an sh-No Target RNA (PRO-NT). Shown are 3 biological replicates for senescent cells expressing No Target or sh- H2AFJ RNAs. Error bars represent mean±s.d. t -tests with * P

    Article Snippet: shRNA knock-down of H2AFJ pTRIPz-sh-No Target (Thermo RHS4743), pTRIPz-sh2-H2AFJ (Thermo V3THS_302395) pTRIPz-sh3-H2AFJ (Thermo V3THS_351902), and pGIPz-sh-NoTarget (RHS4346) were purchased from Thermo Scientific.

    Techniques: Expressing, Quantitative RT-PCR

    The H2A.J C-terminal sequence is required for its function. WI-38 hTERT cells were infected with pGIPz lentiviruses expressing a No Target shRNA (sh-NT) or an sh3- H2AFJ RNA. The sh3- H2AFJ cell line was super-infected with pTRIPz lentiviruses expressing sh3-resistant H2AFJ cDNAs encoding WT-H2A.J or a mutant H2A.J in which the C-terminus of H2A.J was substituted with the C terminus of canonical H2A type 1 (H2A.J-Cter-H2A). Both the WT and mutant H2A.J contained an N-terminal Flag-HA tag. RNA was isolated from cells induced into senescence by etoposide treatment for 3 weeks and compared to levels in proliferating sh-No Target cells. ( a ) Schematic of V11 and C-terminal aa sequences of H2A.J and H2A.J-C-ter-H2A. ( b ) RT–qPCR quantification of inflammatory gene expression for three biological replicates (independent cultures at different dates) for the indicated genes. The induction ratio for these genes in senescent versus proliferating sh-NT cells varied between biological replicates. The extent to which ectopic WT-H2A.J restored defective gene expression in the sh- H2AFJ knockdown cells also varied between biological replicates. However, the H2A.J-Cter-H2A mutant was ineffective at restoring inflammatory gene expression, indicating the functional importance of the specific H2A.J C-terminal sequence. For each biological replicate, the mean and s.d. are shown for three qPCR technical replicates.

    Journal: Nature Communications

    Article Title: Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression

    doi: 10.1038/ncomms14995

    Figure Lengend Snippet: The H2A.J C-terminal sequence is required for its function. WI-38 hTERT cells were infected with pGIPz lentiviruses expressing a No Target shRNA (sh-NT) or an sh3- H2AFJ RNA. The sh3- H2AFJ cell line was super-infected with pTRIPz lentiviruses expressing sh3-resistant H2AFJ cDNAs encoding WT-H2A.J or a mutant H2A.J in which the C-terminus of H2A.J was substituted with the C terminus of canonical H2A type 1 (H2A.J-Cter-H2A). Both the WT and mutant H2A.J contained an N-terminal Flag-HA tag. RNA was isolated from cells induced into senescence by etoposide treatment for 3 weeks and compared to levels in proliferating sh-No Target cells. ( a ) Schematic of V11 and C-terminal aa sequences of H2A.J and H2A.J-C-ter-H2A. ( b ) RT–qPCR quantification of inflammatory gene expression for three biological replicates (independent cultures at different dates) for the indicated genes. The induction ratio for these genes in senescent versus proliferating sh-NT cells varied between biological replicates. The extent to which ectopic WT-H2A.J restored defective gene expression in the sh- H2AFJ knockdown cells also varied between biological replicates. However, the H2A.J-Cter-H2A mutant was ineffective at restoring inflammatory gene expression, indicating the functional importance of the specific H2A.J C-terminal sequence. For each biological replicate, the mean and s.d. are shown for three qPCR technical replicates.

    Article Snippet: shRNA knock-down of H2AFJ pTRIPz-sh-No Target (Thermo RHS4743), pTRIPz-sh2-H2AFJ (Thermo V3THS_302395) pTRIPz-sh3-H2AFJ (Thermo V3THS_351902), and pGIPz-sh-NoTarget (RHS4346) were purchased from Thermo Scientific.

    Techniques: Sequencing, Infection, Expressing, shRNA, Mutagenesis, Isolation, Quantitative RT-PCR, Functional Assay, Real-time Polymerase Chain Reaction

    ChIP of H2A.J on selected SASP and non-SASP genes. CCL2, IL1B , and CXCL5 (SASP) and GAPDH, BCL2A1 (non-SASP) genes were selected for targeted ChIP from cells in proliferation or etoposide-induced senescence. H2A.J was immunoprecipitated from sonicated crosslinked chromatin from WI38hTERT fibroblasts expressing an sh-NoTarget RNA (NT) or an sh3- H2AFJ RNA (shJ). The red rectangles indicate the position of the primers used for determining the percentage of DNA immunoprecipitated relative to the input. The sites chosen in the promoter regions of the SASP genes contain NF-kB binding motifs. The scale bars below the figures show gene expression heat maps indicating that H2A.J contributes to the increased expression of these SASP genes in senescence. GAPDH shows relatively uniformly high expression in proliferating and senescent cells whereas BCL2A1 shows relatively uniformly low expression. The percent input is shown as the mean±s.d. of 3 biological replicates.

    Journal: Nature Communications

    Article Title: Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression

    doi: 10.1038/ncomms14995

    Figure Lengend Snippet: ChIP of H2A.J on selected SASP and non-SASP genes. CCL2, IL1B , and CXCL5 (SASP) and GAPDH, BCL2A1 (non-SASP) genes were selected for targeted ChIP from cells in proliferation or etoposide-induced senescence. H2A.J was immunoprecipitated from sonicated crosslinked chromatin from WI38hTERT fibroblasts expressing an sh-NoTarget RNA (NT) or an sh3- H2AFJ RNA (shJ). The red rectangles indicate the position of the primers used for determining the percentage of DNA immunoprecipitated relative to the input. The sites chosen in the promoter regions of the SASP genes contain NF-kB binding motifs. The scale bars below the figures show gene expression heat maps indicating that H2A.J contributes to the increased expression of these SASP genes in senescence. GAPDH shows relatively uniformly high expression in proliferating and senescent cells whereas BCL2A1 shows relatively uniformly low expression. The percent input is shown as the mean±s.d. of 3 biological replicates.

    Article Snippet: shRNA knock-down of H2AFJ pTRIPz-sh-No Target (Thermo RHS4743), pTRIPz-sh2-H2AFJ (Thermo V3THS_302395) pTRIPz-sh3-H2AFJ (Thermo V3THS_351902), and pGIPz-sh-NoTarget (RHS4346) were purchased from Thermo Scientific.

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Sonication, Expressing, Binding Assay