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  • 99
    Addgene inc plko
    The glycogen synthase kinase 3 (GSK3α/β) isoforms activity inhibits cAMP response element-binding (CREB) phosphorylation at Ser133. Bovine endothelial cells (BECs) were left with medium only or transfected for 120 h with <t>PLKO.1</t> vector containing siRNA sequences to silence the expression of GSK3α and GSK3β genes or with PLKO.1 control vector. (A) Unphosphorylated GSK3α/β was detected to check for protein loading. Error bars in graph represent the mean ± SEM ( n = 3) of three independent experiments. * P
    Plko, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 2906 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Horizon Discovery plko
    The glycogen synthase kinase 3 (GSK3α/β) isoforms activity inhibits cAMP response element-binding (CREB) phosphorylation at Ser133. Bovine endothelial cells (BECs) were left with medium only or transfected for 120 h with <t>PLKO.1</t> vector containing siRNA sequences to silence the expression of GSK3α and GSK3β genes or with PLKO.1 control vector. (A) Unphosphorylated GSK3α/β was detected to check for protein loading. Error bars in graph represent the mean ± SEM ( n = 3) of three independent experiments. * P
    Plko, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 99/100, based on 284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore plko
    (A) Computational analysis of the <t>EGR-1</t> recognition sequence [GCG(G/T)GGCG] in the genomic sequence 1,500 bp upstream and 500 bp downstream of the transcription initiation site of PDGF-A, MIC-1, and FASN. Black vertical lines and black rectangular boxes denote genomic sequences and exons, respectively; vertical arrow heads indicate EGR-1 recognition sequences. (B) EGR-1, PDGF-A, MIC-1, and FASN protein expression in RWPE-1 cells transiently transfected with pcDNA3.1/EGR-1 (EGR-1 overexpression) or <t>pLKO.1/EGR-1</t> shRNA (EGR-1 suppression), and their empty plasmid controls. Double bands in EGR-1 represent post-translational modifications ( 44 ). The fold change difference compared to empty plasmid control and determined by densitometry as a ratio with β-actin signal is indicated in the small bar graphs (left bar, EGR-1 overexpression; right bar, EGR-1 suppression). (C) Relative mRNA expression of PDGF-A, MIC-1, and FASN in RWPE-1 cells transiently transfected with pcDNA3.1/EGR-1 (EGR-1 overexpression) or pLKO.1/EGR-1 shRNA (EGR-1 suppression), and their empty plasmid controls. Bars represent averages of triplicates ± standard deviation; * Statistical significance (p≤0.05) from pcDNA3.1 and pLKO.1 plasmid vector control, respectively. EGR-1, early growth response-1; PDGF-A, platelet-derived growth factor-A; MIC-1, macrophage inhibitory cytokine-1; FASN, fatty acid synthase.
    Plko, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2514 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher plko
    <t>CBF-β</t> is critical for the primary HIV-1 Vif-Cul5 interaction. CBF-β is important for HIV-1 89.6 Vif-mediated (A) and HIV-1 Yu2 Vif-mediated (D) degradation of human A3G. HEK293T cells were transfected with expression vectors for HIV-1 89.6
    Plko, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2059 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    GE Healthcare plko
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore control plko
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Control Plko, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore plko shcdk9
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko Shcdk9, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Cellecta plko 1
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko 1, supplied by Cellecta, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Addgene inc plko 3
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko 3, supplied by Addgene inc, used in various techniques. Bioz Stars score: 99/100, based on 258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore plko shsox2
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko Shsox2, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Addgene inc plko teton
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko Teton, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GenePharma Company plko 1
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko 1, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Thermo Fisher plko background
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko Background, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    tiangen biotech co plko 1
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko 1, supplied by tiangen biotech co, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore plko 3xlaco
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko 3xlaco, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Addgene inc plko shd
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko Shd, supplied by Addgene inc, used in various techniques. Bioz Stars score: 80/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Horizon Discovery plko 1
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko 1, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 92/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Millipore plko p53sh
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko P53sh, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Novartis plko tet on
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko Tet On, supplied by Novartis, used in various techniques. Bioz Stars score: 78/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc plko puro
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko Puro, supplied by Addgene inc, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore plko system
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko System, supplied by Millipore, used in various techniques. Bioz Stars score: 78/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa plko 1
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko 1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher plko shcdk2
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko Shcdk2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Biosettia plko 1
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko 1, supplied by Biosettia, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Thermo Fisher plko trc00010203
    Different apoptotic rate of CNE2-shANXA1 and <t>CNE2-pLKO.1</t> cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using
    Plko Trc00010203, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore plko shecad
    E-Cadherin is required for in vivo growth of SUM149, Mary-X and 4T1 breast cancer cell lines (a) Longitudinal bioluminescent imaging (BLI) of SUM149-shNT and <t>SUM149-shECad</t> cells growth in MFP. Similar results were obtained with SUM149-LUC and SUM149-ZEB1 clones. (b,d) M-SUM149 clones display reduced in vivo growth capacity. Five hundred thousand of E-SUM149 or M-SUM149 clones were implanted into MFP and BLI was measured to monitor tumor growth. BLI was performed on a weekly basis for 8 weeks. Five mice per group was used. Similar results were obtained when 50,000 cells were used (data not shown). (c,e) Reduced tumor volume of M- SUM149 clones when compared to E-SUM149 clones 8 weeks post implantation (n=5). (f,h) Western blot validation of xenograft tissue cell extract from E-SUM149 and M-SUM149 clones. SUM149-shECad tumors maintained their reduced E-Cadherin expression while retaining N-Cadherin expression. Additionally, βcatenin expression levels were correlated with E-Cadherin expression. (g) Based on immunohistochemical staining, reduced E-Cadherin staining was maintained in SUM149-shECad. (i) Immunohistochemical staining of E-Cadherin protein on SUM149-LUC and SUM149-ZEB1 tumor tissues. E-Cadherin expression was retained in the SUM149-ZEB1 tumor tissue similar to the result observed by western blot. (k) BLI of tumor burden in intra-cardiac injection metastatic model. E-Cadherin expression in SUM149 cells is required for metastatic colonization. Longitudinal study of metastatic burden of SUM149-shNT (n=4) and SUM149-shECad (n=4). Decrease metastatic foci in pancreas (l) and liver (m) of SUM149-shECad injected mice. Tumors localization within the tissues are traced in red. (n, left) Western blot showing efficient knockdown of E-Cadherin in 4T1-shECad cells. (n, right) Dramatic reduction of MFP in vivo growth of 4T1-shECad cells compared to either 4T1 parental or control 4T1-shNS. (o, left) Efficient knockdown of E-Cadherin using the modified <t>pLKO-LiP</t> vector backbone. (o, right) Reduced in vivo growth of Mary-X-shEcad-LiP cells compared to Mary-X-shNT-LiP
    Plko Shecad, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore plko shrnas
    E-Cadherin is required for in vivo growth of SUM149, Mary-X and 4T1 breast cancer cell lines (a) Longitudinal bioluminescent imaging (BLI) of SUM149-shNT and <t>SUM149-shECad</t> cells growth in MFP. Similar results were obtained with SUM149-LUC and SUM149-ZEB1 clones. (b,d) M-SUM149 clones display reduced in vivo growth capacity. Five hundred thousand of E-SUM149 or M-SUM149 clones were implanted into MFP and BLI was measured to monitor tumor growth. BLI was performed on a weekly basis for 8 weeks. Five mice per group was used. Similar results were obtained when 50,000 cells were used (data not shown). (c,e) Reduced tumor volume of M- SUM149 clones when compared to E-SUM149 clones 8 weeks post implantation (n=5). (f,h) Western blot validation of xenograft tissue cell extract from E-SUM149 and M-SUM149 clones. SUM149-shECad tumors maintained their reduced E-Cadherin expression while retaining N-Cadherin expression. Additionally, βcatenin expression levels were correlated with E-Cadherin expression. (g) Based on immunohistochemical staining, reduced E-Cadherin staining was maintained in SUM149-shECad. (i) Immunohistochemical staining of E-Cadherin protein on SUM149-LUC and SUM149-ZEB1 tumor tissues. E-Cadherin expression was retained in the SUM149-ZEB1 tumor tissue similar to the result observed by western blot. (k) BLI of tumor burden in intra-cardiac injection metastatic model. E-Cadherin expression in SUM149 cells is required for metastatic colonization. Longitudinal study of metastatic burden of SUM149-shNT (n=4) and SUM149-shECad (n=4). Decrease metastatic foci in pancreas (l) and liver (m) of SUM149-shECad injected mice. Tumors localization within the tissues are traced in red. (n, left) Western blot showing efficient knockdown of E-Cadherin in 4T1-shECad cells. (n, right) Dramatic reduction of MFP in vivo growth of 4T1-shECad cells compared to either 4T1 parental or control 4T1-shNS. (o, left) Efficient knockdown of E-Cadherin using the modified <t>pLKO-LiP</t> vector backbone. (o, right) Reduced in vivo growth of Mary-X-shEcad-LiP cells compared to Mary-X-shNT-LiP
    Plko Shrnas, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore plko plasmid plko 1 vector
    <t>MYB</t> represses the fetal globin gene by upregulating both the DRED and KLF1/BCL11A pathways in human erythroid cells. (A) Expression levels of MYB gene in primary human CD34 + -derived cells transduced with <t>pLKO.1</t> control or pLKO.1 with shRNAs targeting
    Plko Plasmid Plko 1 Vector, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc plko 1 empty
    <t>MYB</t> represses the fetal globin gene by upregulating both the DRED and KLF1/BCL11A pathways in human erythroid cells. (A) Expression levels of MYB gene in primary human CD34 + -derived cells transduced with <t>pLKO.1</t> control or pLKO.1 with shRNAs targeting
    Plko 1 Empty, supplied by Addgene inc, used in various techniques. Bioz Stars score: 89/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore plko 1 puro
    <t>MYB</t> represses the fetal globin gene by upregulating both the DRED and KLF1/BCL11A pathways in human erythroid cells. (A) Expression levels of MYB gene in primary human CD34 + -derived cells transduced with <t>pLKO.1</t> control or pLKO.1 with shRNAs targeting
    Plko 1 Puro, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 315 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    <t>MYB</t> represses the fetal globin gene by upregulating both the DRED and KLF1/BCL11A pathways in human erythroid cells. (A) Expression levels of MYB gene in primary human CD34 + -derived cells transduced with <t>pLKO.1</t> control or pLKO.1 with shRNAs targeting
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    <t>MYB</t> represses the fetal globin gene by upregulating both the DRED and KLF1/BCL11A pathways in human erythroid cells. (A) Expression levels of MYB gene in primary human CD34 + -derived cells transduced with <t>pLKO.1</t> control or pLKO.1 with shRNAs targeting
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    Image Search Results


    The glycogen synthase kinase 3 (GSK3α/β) isoforms activity inhibits cAMP response element-binding (CREB) phosphorylation at Ser133. Bovine endothelial cells (BECs) were left with medium only or transfected for 120 h with PLKO.1 vector containing siRNA sequences to silence the expression of GSK3α and GSK3β genes or with PLKO.1 control vector. (A) Unphosphorylated GSK3α/β was detected to check for protein loading. Error bars in graph represent the mean ± SEM ( n = 3) of three independent experiments. * P

    Journal: Frontiers in Immunology

    Article Title: Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus

    doi: 10.3389/fimmu.2018.00092

    Figure Lengend Snippet: The glycogen synthase kinase 3 (GSK3α/β) isoforms activity inhibits cAMP response element-binding (CREB) phosphorylation at Ser133. Bovine endothelial cells (BECs) were left with medium only or transfected for 120 h with PLKO.1 vector containing siRNA sequences to silence the expression of GSK3α and GSK3β genes or with PLKO.1 control vector. (A) Unphosphorylated GSK3α/β was detected to check for protein loading. Error bars in graph represent the mean ± SEM ( n = 3) of three independent experiments. * P

    Article Snippet: Plasmid pLKO.1-GSK3β-#1 was a gift from Alex Toker (Addgene plasmid #32497) and PLKO.1 plasmid was a gift from Bob Weinberg (Addgene plasmid # 8453). pCF CREB M1 was a gift from Marc Montminy (Addgene plasmid # 22969).

    Techniques: Activity Assay, Binding Assay, Transfection, Plasmid Preparation, Expressing

    MiR-128a overexpression in OCI-AML3 cell line. ( a and b ) qRT-PCR of miR-128a ( a ) and Lin28A ( b ) in OCI-AML3 infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment. ( c and d ) Representative histogram plots of CD11b+ ( c ) and CD14+ cells ( d ) in OCI-AML3 infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment. ( e ) Percentage of CD11b+ and CD14+ OCI-AML3 cells infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment, by cytofluorimetric analysis. ( f ) May–Grünwald Giemsa staining of OCI-AML3 infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment. ( g ) Colony-forming assay of OCI-AML3 after infection with pLKO.1_scr or pLKO.1_miR-128a. Colonies were observed at day 14 of the semisolid culture under × 20 magnification. ( h ) Count of CFU-M colonies. The line and bar graphs represented mean±S.D. from three independent experiments. Statistically significant analyses are indicated by asterisks: * P

    Journal: Cell Death & Disease

    Article Title: Knockdown of miR-128a induces Lin28a expression and reverts myeloid differentiation blockage in acute myeloid leukemia

    doi: 10.1038/cddis.2017.253

    Figure Lengend Snippet: MiR-128a overexpression in OCI-AML3 cell line. ( a and b ) qRT-PCR of miR-128a ( a ) and Lin28A ( b ) in OCI-AML3 infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment. ( c and d ) Representative histogram plots of CD11b+ ( c ) and CD14+ cells ( d ) in OCI-AML3 infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment. ( e ) Percentage of CD11b+ and CD14+ OCI-AML3 cells infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment, by cytofluorimetric analysis. ( f ) May–Grünwald Giemsa staining of OCI-AML3 infected with pLKO.1_scr or pLKO.1_miR-128a after 24, 48 and 72 h of PMA treatment. ( g ) Colony-forming assay of OCI-AML3 after infection with pLKO.1_scr or pLKO.1_miR-128a. Colonies were observed at day 14 of the semisolid culture under × 20 magnification. ( h ) Count of CFU-M colonies. The line and bar graphs represented mean±S.D. from three independent experiments. Statistically significant analyses are indicated by asterisks: * P

    Article Snippet: Lentivirus production and infection MiR-128a expression vector were made by cloning ~60 bp 5′ and 3′ of the pre-miRNA into the multiple cloning site for pLKO.1 (Addgene, Cambridge, MA, USA).

    Techniques: Over Expression, Quantitative RT-PCR, Infection, Staining

    Loss of TNFR1 and SAM68 rescues cellular viability in BRCA2-depleted cancer cells. a Schematic overview of TNFR1 complex formation upon TNFα binding, leading to cell survival (complex I) or delayed caspase activation and cell death (complex II). b Flow cytometry analysis of KBM-7-shBRCA2 #2 cells, additionally carrying indicated shRNA vectors with IRES-driven mCherry cassettes. Cells were treated with doxycycline for 14 days and percentages of mCherry-positive cells were measured. c Indicated KBM-7-shBRCA2 cells carrying mCherry shRNA cassettes targeting TNFR1, SAM68 or a control sequence (‘SCR’) were treated with or without doxycycline to induce BRCA2 shRNA expression. Percentages of mCherry-positive cells were measured every 3 or 4 days for 3 weeks after start of doxycycline treatment. Ratios of mCherry-positive cells in doxycycline treated cultures vs. untreated cultures are indicated. Per condition, at least 30,000 events were measured. d BT-549 cells, stably transduced with pLKO.tet.shBRCA2 #2, were infected with IRES mCherry shRNA vectors as for b . Cells were treated with or without doxycycline, and percentages of mCherry-positive cells were measured. Ratios of mCherry-positive cells at indicated time points vs. mCherry-positive percentages at day 0 are indicated. Error bars indicate s.d. of three independent experiments. P values were calculated using two-tailed Student’s t -test. * P

    Journal: Nature Communications

    Article Title: BRCA2 deficiency instigates cGAS-mediated inflammatory signaling and confers sensitivity to tumor necrosis factor-alpha-mediated cytotoxicity

    doi: 10.1038/s41467-018-07927-y

    Figure Lengend Snippet: Loss of TNFR1 and SAM68 rescues cellular viability in BRCA2-depleted cancer cells. a Schematic overview of TNFR1 complex formation upon TNFα binding, leading to cell survival (complex I) or delayed caspase activation and cell death (complex II). b Flow cytometry analysis of KBM-7-shBRCA2 #2 cells, additionally carrying indicated shRNA vectors with IRES-driven mCherry cassettes. Cells were treated with doxycycline for 14 days and percentages of mCherry-positive cells were measured. c Indicated KBM-7-shBRCA2 cells carrying mCherry shRNA cassettes targeting TNFR1, SAM68 or a control sequence (‘SCR’) were treated with or without doxycycline to induce BRCA2 shRNA expression. Percentages of mCherry-positive cells were measured every 3 or 4 days for 3 weeks after start of doxycycline treatment. Ratios of mCherry-positive cells in doxycycline treated cultures vs. untreated cultures are indicated. Per condition, at least 30,000 events were measured. d BT-549 cells, stably transduced with pLKO.tet.shBRCA2 #2, were infected with IRES mCherry shRNA vectors as for b . Cells were treated with or without doxycycline, and percentages of mCherry-positive cells were measured. Ratios of mCherry-positive cells at indicated time points vs. mCherry-positive percentages at day 0 are indicated. Error bars indicate s.d. of three independent experiments. P values were calculated using two-tailed Student’s t -test. * P

    Article Snippet: Tet-pLKO-puro was a gift from Dmitri Wiederschain (Addgene plasmid #21915) .

    Techniques: Binding Assay, Activation Assay, Flow Cytometry, Cytometry, shRNA, Sequencing, Expressing, Stable Transfection, Transduction, Infection, Two Tailed Test

    (A) Computational analysis of the EGR-1 recognition sequence [GCG(G/T)GGCG] in the genomic sequence 1,500 bp upstream and 500 bp downstream of the transcription initiation site of PDGF-A, MIC-1, and FASN. Black vertical lines and black rectangular boxes denote genomic sequences and exons, respectively; vertical arrow heads indicate EGR-1 recognition sequences. (B) EGR-1, PDGF-A, MIC-1, and FASN protein expression in RWPE-1 cells transiently transfected with pcDNA3.1/EGR-1 (EGR-1 overexpression) or pLKO.1/EGR-1 shRNA (EGR-1 suppression), and their empty plasmid controls. Double bands in EGR-1 represent post-translational modifications ( 44 ). The fold change difference compared to empty plasmid control and determined by densitometry as a ratio with β-actin signal is indicated in the small bar graphs (left bar, EGR-1 overexpression; right bar, EGR-1 suppression). (C) Relative mRNA expression of PDGF-A, MIC-1, and FASN in RWPE-1 cells transiently transfected with pcDNA3.1/EGR-1 (EGR-1 overexpression) or pLKO.1/EGR-1 shRNA (EGR-1 suppression), and their empty plasmid controls. Bars represent averages of triplicates ± standard deviation; * Statistical significance (p≤0.05) from pcDNA3.1 and pLKO.1 plasmid vector control, respectively. EGR-1, early growth response-1; PDGF-A, platelet-derived growth factor-A; MIC-1, macrophage inhibitory cytokine-1; FASN, fatty acid synthase.

    Journal: International Journal of Oncology

    Article Title: Association and regulation of protein factors of field effect in prostate tissues

    doi: 10.3892/ijo.2016.3666

    Figure Lengend Snippet: (A) Computational analysis of the EGR-1 recognition sequence [GCG(G/T)GGCG] in the genomic sequence 1,500 bp upstream and 500 bp downstream of the transcription initiation site of PDGF-A, MIC-1, and FASN. Black vertical lines and black rectangular boxes denote genomic sequences and exons, respectively; vertical arrow heads indicate EGR-1 recognition sequences. (B) EGR-1, PDGF-A, MIC-1, and FASN protein expression in RWPE-1 cells transiently transfected with pcDNA3.1/EGR-1 (EGR-1 overexpression) or pLKO.1/EGR-1 shRNA (EGR-1 suppression), and their empty plasmid controls. Double bands in EGR-1 represent post-translational modifications ( 44 ). The fold change difference compared to empty plasmid control and determined by densitometry as a ratio with β-actin signal is indicated in the small bar graphs (left bar, EGR-1 overexpression; right bar, EGR-1 suppression). (C) Relative mRNA expression of PDGF-A, MIC-1, and FASN in RWPE-1 cells transiently transfected with pcDNA3.1/EGR-1 (EGR-1 overexpression) or pLKO.1/EGR-1 shRNA (EGR-1 suppression), and their empty plasmid controls. Bars represent averages of triplicates ± standard deviation; * Statistical significance (p≤0.05) from pcDNA3.1 and pLKO.1 plasmid vector control, respectively. EGR-1, early growth response-1; PDGF-A, platelet-derived growth factor-A; MIC-1, macrophage inhibitory cytokine-1; FASN, fatty acid synthase.

    Article Snippet: Trypsin-EDTA at 0.25% was used to detach the cells for splitting and reculturing. pcDNA3.1 control and pcDNA3.1/EGR-1 plasmids were a kind gift of Dr W. Xiao (University of Science and Technology of China, Hefei, China). pLKO.1 control and pLKO.1/EGR-1 shRNA plasmids were from Sigma (St. Louis, MO, USA).

    Techniques: Sequencing, Genomic Sequencing, Expressing, Transfection, Over Expression, shRNA, Plasmid Preparation, Standard Deviation, Derivative Assay

    Identification of regulators of stem cell homeostasis in a MuSC proteome-based shRNA screen. ( a ) Mass spectrometry (MS)-based identification of MuSC-enriched proteins (Proteome SC) using samples from fractionated skeletal muscles including purified myotubes, Pax7-GFP − mononuclear cells, percoll-gradient purified and Pax7-GFP + MuSC. ( b ) Immunofluorescence validation of identified proteins (red) counterstained with Pax7 antibody (green) and DAPI (blue; scale bar, 20 μm). ( c ) Schematic outline of the shRNA screen against corresponding genes of the satellite cell proteome. Phenotypic scores are calculated as ratios of Pax7 + /DAPI + nuclei for each well. ( d ) Poisson distribution of relative Pax7 expression for all 2,226 shRNAs targeting 419 genes. Red and green lines indicate phenotypic scores lower or higher than 0.25 percentiles normalized to plko.1 empty vector control. Knockdown of Pax7 reduces proliferation and enhances differentiation of MuSC 6 , whereas knockdown of Nf1 increases the numbers of Pax7 expressing cells 51 . ( e ) shRNA knockdown identifies 90 and 30 candidate genes causing down- and upregulation of Pax7 + /DAPI + ratios, respectively. P.i., post infection; Pos, positive.

    Journal: Nature Communications

    Article Title: Prmt5 is a regulator of muscle stem cell expansion in adult mice

    doi: 10.1038/ncomms8140

    Figure Lengend Snippet: Identification of regulators of stem cell homeostasis in a MuSC proteome-based shRNA screen. ( a ) Mass spectrometry (MS)-based identification of MuSC-enriched proteins (Proteome SC) using samples from fractionated skeletal muscles including purified myotubes, Pax7-GFP − mononuclear cells, percoll-gradient purified and Pax7-GFP + MuSC. ( b ) Immunofluorescence validation of identified proteins (red) counterstained with Pax7 antibody (green) and DAPI (blue; scale bar, 20 μm). ( c ) Schematic outline of the shRNA screen against corresponding genes of the satellite cell proteome. Phenotypic scores are calculated as ratios of Pax7 + /DAPI + nuclei for each well. ( d ) Poisson distribution of relative Pax7 expression for all 2,226 shRNAs targeting 419 genes. Red and green lines indicate phenotypic scores lower or higher than 0.25 percentiles normalized to plko.1 empty vector control. Knockdown of Pax7 reduces proliferation and enhances differentiation of MuSC 6 , whereas knockdown of Nf1 increases the numbers of Pax7 expressing cells 51 . ( e ) shRNA knockdown identifies 90 and 30 candidate genes causing down- and upregulation of Pax7 + /DAPI + ratios, respectively. P.i., post infection; Pos, positive.

    Article Snippet: Lentiviruses overexpressing the coding region of human Prmt5 were generated with a modified lentiviral vector derived from plko.1 (Sigma-Aldrich) in HEK293T cells using the helper plasmids pMD2.G and psPAX2, and used for infection of MuSC.

    Techniques: shRNA, Mass Spectrometry, Purification, Immunofluorescence, Expressing, Plasmid Preparation, Infection

    Effect of FOS, FOSB, and FOSL1 shRNAs on trophoblast cell proliferation. Swan 71 trophoblast cells were infected with lentiviruses carrying pLKO.1 plasmids encoding shRNAs targeting FOS , FOSB , or FOSL1. A , protein expression of FOS ( top ), FOSB ( middle

    Journal: The Journal of Biological Chemistry

    Article Title: The FOS Transcription Factor Family Differentially Controls Trophoblast Migration and Invasion *

    doi: 10.1074/jbc.M113.523746

    Figure Lengend Snippet: Effect of FOS, FOSB, and FOSL1 shRNAs on trophoblast cell proliferation. Swan 71 trophoblast cells were infected with lentiviruses carrying pLKO.1 plasmids encoding shRNAs targeting FOS , FOSB , or FOSL1. A , protein expression of FOS ( top ), FOSB ( middle

    Article Snippet: FOS , FOSB , and FOSL1 shRNA constructs in pLKO.1 vectors were obtained from Sigma-Aldrich.

    Techniques: Infection, Expressing

    CBF-β is critical for the primary HIV-1 Vif-Cul5 interaction. CBF-β is important for HIV-1 89.6 Vif-mediated (A) and HIV-1 Yu2 Vif-mediated (D) degradation of human A3G. HEK293T cells were transfected with expression vectors for HIV-1 89.6

    Journal: Journal of Virology

    Article Title: Evolutionarily Conserved Requirement for Core Binding Factor Beta in the Assembly of the Human Immunodeficiency Virus/Simian Immunodeficiency Virus Vif-Cullin 5-RING E3 Ubiquitin Ligase

    doi: 10.1128/JVI.03833-13

    Figure Lengend Snippet: CBF-β is critical for the primary HIV-1 Vif-Cul5 interaction. CBF-β is important for HIV-1 89.6 Vif-mediated (A) and HIV-1 Yu2 Vif-mediated (D) degradation of human A3G. HEK293T cells were transfected with expression vectors for HIV-1 89.6

    Article Snippet: HEK293T cells were cotransfected with pLKO.1 or pLKO.1–CBF-β (clone TRCN0000016645, 5′-GAAGATAGAGACAGGTCTCAT-3′ [Open Biosystems]) together with pRSV-Rev (where RSV is Rous sarcoma virus), pMDLg/pRRE (where RRE is Rev-responsive element), and pCMV-VSVG (where CMV and VSVG are cytomegalovirus and vesicular stomatitis virus G protein, respectively).

    Techniques: Transfection, Expressing

    CBF-β enhances the interaction of HIV-1 Vif and Cul5 in vitro . (A) Efficient pulldown of Vif–CBF-β–ElonginB/C but not Vif-ElonginB/C by Cul5N-GST. Cul5N-GST, Vif–CBF-β–ElonginB/C, and Vif-ElonginB/C

    Journal: Journal of Virology

    Article Title: Evolutionarily Conserved Requirement for Core Binding Factor Beta in the Assembly of the Human Immunodeficiency Virus/Simian Immunodeficiency Virus Vif-Cullin 5-RING E3 Ubiquitin Ligase

    doi: 10.1128/JVI.03833-13

    Figure Lengend Snippet: CBF-β enhances the interaction of HIV-1 Vif and Cul5 in vitro . (A) Efficient pulldown of Vif–CBF-β–ElonginB/C but not Vif-ElonginB/C by Cul5N-GST. Cul5N-GST, Vif–CBF-β–ElonginB/C, and Vif-ElonginB/C

    Article Snippet: HEK293T cells were cotransfected with pLKO.1 or pLKO.1–CBF-β (clone TRCN0000016645, 5′-GAAGATAGAGACAGGTCTCAT-3′ [Open Biosystems]) together with pRSV-Rev (where RSV is Rous sarcoma virus), pMDLg/pRRE (where RRE is Rev-responsive element), and pCMV-VSVG (where CMV and VSVG are cytomegalovirus and vesicular stomatitis virus G protein, respectively).

    Techniques: In Vitro

    CBF-β from multiple animal species can support HIV-1 Vif function. (A) CBF-β-silenced HEK293T cells were cotransfected with expression vectors for human A3G-HA, HIV-1 Vif, and CBF-β–myc from different animal species as

    Journal: Journal of Virology

    Article Title: Evolutionarily Conserved Requirement for Core Binding Factor Beta in the Assembly of the Human Immunodeficiency Virus/Simian Immunodeficiency Virus Vif-Cullin 5-RING E3 Ubiquitin Ligase

    doi: 10.1128/JVI.03833-13

    Figure Lengend Snippet: CBF-β from multiple animal species can support HIV-1 Vif function. (A) CBF-β-silenced HEK293T cells were cotransfected with expression vectors for human A3G-HA, HIV-1 Vif, and CBF-β–myc from different animal species as

    Article Snippet: HEK293T cells were cotransfected with pLKO.1 or pLKO.1–CBF-β (clone TRCN0000016645, 5′-GAAGATAGAGACAGGTCTCAT-3′ [Open Biosystems]) together with pRSV-Rev (where RSV is Rous sarcoma virus), pMDLg/pRRE (where RRE is Rev-responsive element), and pCMV-VSVG (where CMV and VSVG are cytomegalovirus and vesicular stomatitis virus G protein, respectively).

    Techniques: Expressing

    Vif-Cul5-ElonginB/C forms stable complexes only in the presence of CBF-β. Purified Cul5N (residues 1 to 393) was mixed with purified Vif-ElonginB/C or Vif–CBF-β–ElonginB/C at a molar ratio of 1:1 and incubated at 4°C

    Journal: Journal of Virology

    Article Title: Evolutionarily Conserved Requirement for Core Binding Factor Beta in the Assembly of the Human Immunodeficiency Virus/Simian Immunodeficiency Virus Vif-Cullin 5-RING E3 Ubiquitin Ligase

    doi: 10.1128/JVI.03833-13

    Figure Lengend Snippet: Vif-Cul5-ElonginB/C forms stable complexes only in the presence of CBF-β. Purified Cul5N (residues 1 to 393) was mixed with purified Vif-ElonginB/C or Vif–CBF-β–ElonginB/C at a molar ratio of 1:1 and incubated at 4°C

    Article Snippet: HEK293T cells were cotransfected with pLKO.1 or pLKO.1–CBF-β (clone TRCN0000016645, 5′-GAAGATAGAGACAGGTCTCAT-3′ [Open Biosystems]) together with pRSV-Rev (where RSV is Rous sarcoma virus), pMDLg/pRRE (where RRE is Rev-responsive element), and pCMV-VSVG (where CMV and VSVG are cytomegalovirus and vesicular stomatitis virus G protein, respectively).

    Techniques: Purification, Incubation

    Separate roles for CBF-β in promoting HIV-1 Vif-CRL5 assembly and Vif stability. (A) The proposed mechanisms by which CBF-β functions in Vif regulation. CBF-β could be involved either in Vif-CRL5 E3 ubiquitin (Ub) ligase complex

    Journal: Journal of Virology

    Article Title: Evolutionarily Conserved Requirement for Core Binding Factor Beta in the Assembly of the Human Immunodeficiency Virus/Simian Immunodeficiency Virus Vif-Cullin 5-RING E3 Ubiquitin Ligase

    doi: 10.1128/JVI.03833-13

    Figure Lengend Snippet: Separate roles for CBF-β in promoting HIV-1 Vif-CRL5 assembly and Vif stability. (A) The proposed mechanisms by which CBF-β functions in Vif regulation. CBF-β could be involved either in Vif-CRL5 E3 ubiquitin (Ub) ligase complex

    Article Snippet: HEK293T cells were cotransfected with pLKO.1 or pLKO.1–CBF-β (clone TRCN0000016645, 5′-GAAGATAGAGACAGGTCTCAT-3′ [Open Biosystems]) together with pRSV-Rev (where RSV is Rous sarcoma virus), pMDLg/pRRE (where RRE is Rev-responsive element), and pCMV-VSVG (where CMV and VSVG are cytomegalovirus and vesicular stomatitis virus G protein, respectively).

    Techniques:

    CBF-β is important for SIV Vif but not FIV or BIV Vif function. (A to C) SIVmac Vif-mediated downregulation of RhA3 requires CBF-β. HEK293T cells or CBF-β-silenced HEK293T cells were cotransfected with expression vectors for RhA3G-myc

    Journal: Journal of Virology

    Article Title: Evolutionarily Conserved Requirement for Core Binding Factor Beta in the Assembly of the Human Immunodeficiency Virus/Simian Immunodeficiency Virus Vif-Cullin 5-RING E3 Ubiquitin Ligase

    doi: 10.1128/JVI.03833-13

    Figure Lengend Snippet: CBF-β is important for SIV Vif but not FIV or BIV Vif function. (A to C) SIVmac Vif-mediated downregulation of RhA3 requires CBF-β. HEK293T cells or CBF-β-silenced HEK293T cells were cotransfected with expression vectors for RhA3G-myc

    Article Snippet: HEK293T cells were cotransfected with pLKO.1 or pLKO.1–CBF-β (clone TRCN0000016645, 5′-GAAGATAGAGACAGGTCTCAT-3′ [Open Biosystems]) together with pRSV-Rev (where RSV is Rous sarcoma virus), pMDLg/pRRE (where RRE is Rev-responsive element), and pCMV-VSVG (where CMV and VSVG are cytomegalovirus and vesicular stomatitis virus G protein, respectively).

    Techniques: Expressing

    Rac1 knock-down impairs growth and induces apoptosis in the osteoblast cell line OP9. Photomicrograph of OP9 cells infected with nontargeting virus pLKO.1 (SCR; A) or infected with Rac1-shRNA 88 (pLKO.1 vector) demonstrating cytoskeletal elongation (B

    Journal: Blood

    Article Title: Rac signaling in osteoblastic cells is required for normal bone development but is dispensable for hematopoietic development

    doi: 10.1182/blood-2011-07-368753

    Figure Lengend Snippet: Rac1 knock-down impairs growth and induces apoptosis in the osteoblast cell line OP9. Photomicrograph of OP9 cells infected with nontargeting virus pLKO.1 (SCR; A) or infected with Rac1-shRNA 88 (pLKO.1 vector) demonstrating cytoskeletal elongation (B

    Article Snippet: shRNAs targeting Rac1 were obtained in the pLKO.1 backbone (Open Biosystems) and validated to determine effective gene silencing (coded 88 and 92).

    Techniques: Infection, shRNA, Plasmid Preparation

    Different apoptotic rate of CNE2-shANXA1 and CNE2-pLKO.1 cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using

    Journal: Oncology Letters

    Article Title: Downregulation of Annexin A1 is correlated with radioresistance in nasopharyngeal carcinoma

    doi: 10.3892/ol.2016.5324

    Figure Lengend Snippet: Different apoptotic rate of CNE2-shANXA1 and CNE2-pLKO.1 cells with radiation. (A) Hoechst 33258 staining of apoptotic cells. CNE2-shANXA1 and CNE2-pLKO.1 cells were exposed to 5-Gy irradiation, incubated for 72 h and then apoptosis was assessed using

    Article Snippet: The ANXA1-specific shRNA plasmid, pLKO.1-ANXA1-shRNAs, and empty vector, pLKO.1, were purchased from GE Healthcare Life Sciences (Shanghai, China).

    Techniques: Staining, Irradiation, Incubation

    Establishment of the CNE2-shANXA1 cell line with knockdown of ANXA1. (A) Western blotting was used to detect the expression levels of ANXA1 in the untransfected (control 1), empty vector pLKO.1-transfected (control 2) and pLKO.1-ANXA1-shRNA-tansfected

    Journal: Oncology Letters

    Article Title: Downregulation of Annexin A1 is correlated with radioresistance in nasopharyngeal carcinoma

    doi: 10.3892/ol.2016.5324

    Figure Lengend Snippet: Establishment of the CNE2-shANXA1 cell line with knockdown of ANXA1. (A) Western blotting was used to detect the expression levels of ANXA1 in the untransfected (control 1), empty vector pLKO.1-transfected (control 2) and pLKO.1-ANXA1-shRNA-tansfected

    Article Snippet: The ANXA1-specific shRNA plasmid, pLKO.1-ANXA1-shRNAs, and empty vector, pLKO.1, were purchased from GE Healthcare Life Sciences (Shanghai, China).

    Techniques: Western Blot, Expressing, Plasmid Preparation, Transfection, shRNA

    Different sensitivity to radiation in CNE2-shANXA1 and CNE2-pLKO.1 cells. (A and B) Clonogenic survival assay. CNE2-shANXA1 and CNE2-pLKO.1 cells plated onto six-well culture plates were irradiated with a range of radiation doses (0–8 Gy), and

    Journal: Oncology Letters

    Article Title: Downregulation of Annexin A1 is correlated with radioresistance in nasopharyngeal carcinoma

    doi: 10.3892/ol.2016.5324

    Figure Lengend Snippet: Different sensitivity to radiation in CNE2-shANXA1 and CNE2-pLKO.1 cells. (A and B) Clonogenic survival assay. CNE2-shANXA1 and CNE2-pLKO.1 cells plated onto six-well culture plates were irradiated with a range of radiation doses (0–8 Gy), and

    Article Snippet: The ANXA1-specific shRNA plasmid, pLKO.1-ANXA1-shRNAs, and empty vector, pLKO.1, were purchased from GE Healthcare Life Sciences (Shanghai, China).

    Techniques: Clonogenic Cell Survival Assay, Irradiation

    E-Cadherin is required for in vivo growth of SUM149, Mary-X and 4T1 breast cancer cell lines (a) Longitudinal bioluminescent imaging (BLI) of SUM149-shNT and SUM149-shECad cells growth in MFP. Similar results were obtained with SUM149-LUC and SUM149-ZEB1 clones. (b,d) M-SUM149 clones display reduced in vivo growth capacity. Five hundred thousand of E-SUM149 or M-SUM149 clones were implanted into MFP and BLI was measured to monitor tumor growth. BLI was performed on a weekly basis for 8 weeks. Five mice per group was used. Similar results were obtained when 50,000 cells were used (data not shown). (c,e) Reduced tumor volume of M- SUM149 clones when compared to E-SUM149 clones 8 weeks post implantation (n=5). (f,h) Western blot validation of xenograft tissue cell extract from E-SUM149 and M-SUM149 clones. SUM149-shECad tumors maintained their reduced E-Cadherin expression while retaining N-Cadherin expression. Additionally, βcatenin expression levels were correlated with E-Cadherin expression. (g) Based on immunohistochemical staining, reduced E-Cadherin staining was maintained in SUM149-shECad. (i) Immunohistochemical staining of E-Cadherin protein on SUM149-LUC and SUM149-ZEB1 tumor tissues. E-Cadherin expression was retained in the SUM149-ZEB1 tumor tissue similar to the result observed by western blot. (k) BLI of tumor burden in intra-cardiac injection metastatic model. E-Cadherin expression in SUM149 cells is required for metastatic colonization. Longitudinal study of metastatic burden of SUM149-shNT (n=4) and SUM149-shECad (n=4). Decrease metastatic foci in pancreas (l) and liver (m) of SUM149-shECad injected mice. Tumors localization within the tissues are traced in red. (n, left) Western blot showing efficient knockdown of E-Cadherin in 4T1-shECad cells. (n, right) Dramatic reduction of MFP in vivo growth of 4T1-shECad cells compared to either 4T1 parental or control 4T1-shNS. (o, left) Efficient knockdown of E-Cadherin using the modified pLKO-LiP vector backbone. (o, right) Reduced in vivo growth of Mary-X-shEcad-LiP cells compared to Mary-X-shNT-LiP

    Journal: Oncotarget

    Article Title: The Paradox of E-Cadherin: Role in response to hypoxia in the tumor microenvironment and regulation of energy metabolism

    doi:

    Figure Lengend Snippet: E-Cadherin is required for in vivo growth of SUM149, Mary-X and 4T1 breast cancer cell lines (a) Longitudinal bioluminescent imaging (BLI) of SUM149-shNT and SUM149-shECad cells growth in MFP. Similar results were obtained with SUM149-LUC and SUM149-ZEB1 clones. (b,d) M-SUM149 clones display reduced in vivo growth capacity. Five hundred thousand of E-SUM149 or M-SUM149 clones were implanted into MFP and BLI was measured to monitor tumor growth. BLI was performed on a weekly basis for 8 weeks. Five mice per group was used. Similar results were obtained when 50,000 cells were used (data not shown). (c,e) Reduced tumor volume of M- SUM149 clones when compared to E-SUM149 clones 8 weeks post implantation (n=5). (f,h) Western blot validation of xenograft tissue cell extract from E-SUM149 and M-SUM149 clones. SUM149-shECad tumors maintained their reduced E-Cadherin expression while retaining N-Cadherin expression. Additionally, βcatenin expression levels were correlated with E-Cadherin expression. (g) Based on immunohistochemical staining, reduced E-Cadherin staining was maintained in SUM149-shECad. (i) Immunohistochemical staining of E-Cadherin protein on SUM149-LUC and SUM149-ZEB1 tumor tissues. E-Cadherin expression was retained in the SUM149-ZEB1 tumor tissue similar to the result observed by western blot. (k) BLI of tumor burden in intra-cardiac injection metastatic model. E-Cadherin expression in SUM149 cells is required for metastatic colonization. Longitudinal study of metastatic burden of SUM149-shNT (n=4) and SUM149-shECad (n=4). Decrease metastatic foci in pancreas (l) and liver (m) of SUM149-shECad injected mice. Tumors localization within the tissues are traced in red. (n, left) Western blot showing efficient knockdown of E-Cadherin in 4T1-shECad cells. (n, right) Dramatic reduction of MFP in vivo growth of 4T1-shECad cells compared to either 4T1 parental or control 4T1-shNS. (o, left) Efficient knockdown of E-Cadherin using the modified pLKO-LiP vector backbone. (o, right) Reduced in vivo growth of Mary-X-shEcad-LiP cells compared to Mary-X-shNT-LiP

    Article Snippet: The PCR product was gel purified and digested with BamHI and ligated to BamHI-digested pLKO-NT (Sigma; SHC002) and pLKO-shECad (Sigma; TRC0000039666) to generate the pLKO-shNT-LiP and pLKO-shECad-LiP plasmids respectively.

    Techniques: In Vivo, Imaging, Clone Assay, Mouse Assay, Western Blot, Expressing, Immunohistochemistry, Staining, Injection, Modification, Plasmid Preparation

    MYB represses the fetal globin gene by upregulating both the DRED and KLF1/BCL11A pathways in human erythroid cells. (A) Expression levels of MYB gene in primary human CD34 + -derived cells transduced with pLKO.1 control or pLKO.1 with shRNAs targeting

    Journal: Molecular and Cellular Biology

    Article Title: Disruption of the Hbs1l-Myb Locus Causes Hereditary Persistence of Fetal Hemoglobin in a Mouse Model

    doi: 10.1128/MCB.01617-12

    Figure Lengend Snippet: MYB represses the fetal globin gene by upregulating both the DRED and KLF1/BCL11A pathways in human erythroid cells. (A) Expression levels of MYB gene in primary human CD34 + -derived cells transduced with pLKO.1 control or pLKO.1 with shRNAs targeting

    Article Snippet: Lentiviral constructs for expression of short hairpin RNA (shRNA) targeting human MYB in the pLKO vector (TRCN0000040058 and TRCN0000009853) were obtained from Sigma-Aldrich.

    Techniques: Expressing, Derivative Assay, Transduction