Plasmid Preparation:
Article Title: Autocrine production of IL-11 mediates tumorigenicity in hypoxic cancer cells
Article Snippet: Anti–HIF-1a monoclonal antibody was from Novus Biologicals, and anti–c-Fos polyclonal antibody was from Transient transfection and luciferase activity. .. PC3 cells were seeded in 48-well plates (15,000 cells per well) and transiently transfected with luciferase reporter plasmids containing different fragments of the IL11 promoter either with an irrelevant DNA plasmid or with a vector expressing a stable form of HIF-1a (HA-HIF1a P402A/P564A-pBabe-puro, plasmid 19005; Addgene) (61), using FuGene6 (Roche Applied Science) according to the manufacturer’s instructions. .. Twenty-four hours following transfection, cells were cultured under normoxic or hypoxic conditions for an additional 24 hours and luciferase levels were measured using the Luciferase Assay System with Reporter Lysis Buffer kit (Promega).
Article Title: Hypoxia-induced CTCF promotes EMT in breast cancer.
Article Snippet: .. For manipulation of HIF-1a expression in dCa9-TET1 transfected normoxic cells, we transfect these cells with a plasmid constitutively expressing a stable form of HIF-1a (HA-HIF1alpha P402A/P564A-pBabe-puro), which was a gift from William Kaelin (Addgene plasmid # 19005; http://n2t.net/addgene:19005; RRID: Addgene_19005,26). ..
Article Title: Hypoxia-induced CTCF promotes EMT in breast cancer.
Article Snippet: To induce varying degree of CTCF downregulation under hypoxia, different concentration of doxycycline (Sigma, D3072) was added in the cell culture medium and cells were kept in hypoxia. .. For stable HIF-1a expression in normoxic cells, we transfectDNMT3A Knockdown cells with a plasmid constitutively expressing a stable form of HIF-1a (HA-HIF1alpha P402A/P564A-pBabe-puro), which was a gift from William Kaelin (Addgene plasmid # 19005; http://n2t.net/addgene:19005; RRID: Addgene_19005,26). sgRNA designing and cloning in dCas9-DNMT3A or dCas9-TET1 expression vector and cell transfections Guide sequences targeting the loci of interest were selected using the GT-Scan web-tool (http://gt-scan.braembl.org.au/gt-scan/,39) and CRISPick (https://portals.broadinstitute. org/gppx/crispick/public40). .. All sgRNAwere synthesized by oligo annealing and cloned into dCas9-DNMT3A (Addgene #74407) or dCas9-TET1 (Addgene #83340) expression vectors via BbsI (Fermentas, Cat No. ER1012) sites as described previously.25,27 Additionally, non-targeting (NT) control guide RNAs (sgcontrol) was also cloned into dCas9-TET1 (Addgene #83340) expression vector and Addgene #71830 having non-targeting (NT) control guide RNAs (sgcontrol) was used for dCas9-DNMT3A system.
Transfection:Article Title: Autocrine production of IL-11 mediates tumorigenicity in hypoxic cancer cells
Article Snippet: Anti–HIF-1a monoclonal antibody was from Novus Biologicals, and anti–c-Fos polyclonal antibody was from Transient transfection and luciferase activity. .. PC3 cells were seeded in 48-well plates (15,000 cells per well) and transiently transfected with luciferase reporter plasmids containing different fragments of the IL11 promoter either with an irrelevant DNA plasmid or with a vector expressing a stable form of HIF-1a (HA-HIF1a P402A/P564A-pBabe-puro, plasmid 19005; Addgene) (61), using FuGene6 (Roche Applied Science) according to the manufacturer’s instructions. .. Twenty-four hours following transfection, cells were cultured under normoxic or hypoxic conditions for an additional 24 hours and luciferase levels were measured using the Luciferase Assay System with Reporter Lysis Buffer kit (Promega).
Article Title: Hypoxia-induced CTCF promotes EMT in breast cancer.
Article Snippet: .. For manipulation of HIF-1a expression in dCa9-TET1 transfected normoxic cells, we transfect these cells with a plasmid constitutively expressing a stable form of HIF-1a (HA-HIF1alpha P402A/P564A-pBabe-puro), which was a gift from William Kaelin (Addgene plasmid # 19005; http://n2t.net/addgene:19005; RRID: Addgene_19005,26). ..
Article Title: Hypoxia-induced CTCF promotes EMT in breast cancer.
Article Snippet: To induce varying degree of CTCF downregulation under hypoxia, different concentration of doxycycline (Sigma, D3072) was added in the cell culture medium and cells were kept in hypoxia. .. For stable HIF-1a expression in normoxic cells, we transfectDNMT3A Knockdown cells with a plasmid constitutively expressing a stable form of HIF-1a (HA-HIF1alpha P402A/P564A-pBabe-puro), which was a gift from William Kaelin (Addgene plasmid # 19005; http://n2t.net/addgene:19005; RRID: Addgene_19005,26). sgRNA designing and cloning in dCas9-DNMT3A or dCas9-TET1 expression vector and cell transfections Guide sequences targeting the loci of interest were selected using the GT-Scan web-tool (http://gt-scan.braembl.org.au/gt-scan/,39) and CRISPick (https://portals.broadinstitute. org/gppx/crispick/public40). .. All sgRNAwere synthesized by oligo annealing and cloned into dCas9-DNMT3A (Addgene #74407) or dCas9-TET1 (Addgene #83340) expression vectors via BbsI (Fermentas, Cat No. ER1012) sites as described previously.25,27 Additionally, non-targeting (NT) control guide RNAs (sgcontrol) was also cloned into dCas9-TET1 (Addgene #83340) expression vector and Addgene #71830 having non-targeting (NT) control guide RNAs (sgcontrol) was used for dCas9-DNMT3A system.
Luciferase:Article Title: Autocrine production of IL-11 mediates tumorigenicity in hypoxic cancer cells
Article Snippet: Anti–HIF-1a monoclonal antibody was from Novus Biologicals, and anti–c-Fos polyclonal antibody was from Transient transfection and luciferase activity. .. PC3 cells were seeded in 48-well plates (15,000 cells per well) and transiently transfected with luciferase reporter plasmids containing different fragments of the IL11 promoter either with an irrelevant DNA plasmid or with a vector expressing a stable form of HIF-1a (HA-HIF1a P402A/P564A-pBabe-puro, plasmid 19005; Addgene) (61), using FuGene6 (Roche Applied Science) according to the manufacturer’s instructions. .. Twenty-four hours following transfection, cells were cultured under normoxic or hypoxic conditions for an additional 24 hours and luciferase levels were measured using the Luciferase Assay System with Reporter Lysis Buffer kit (Promega).
Expressing:Article Title: Autocrine production of IL-11 mediates tumorigenicity in hypoxic cancer cells
Article Snippet: Anti–HIF-1a monoclonal antibody was from Novus Biologicals, and anti–c-Fos polyclonal antibody was from Transient transfection and luciferase activity. .. PC3 cells were seeded in 48-well plates (15,000 cells per well) and transiently transfected with luciferase reporter plasmids containing different fragments of the IL11 promoter either with an irrelevant DNA plasmid or with a vector expressing a stable form of HIF-1a (HA-HIF1a P402A/P564A-pBabe-puro, plasmid 19005; Addgene) (61), using FuGene6 (Roche Applied Science) according to the manufacturer’s instructions. .. Twenty-four hours following transfection, cells were cultured under normoxic or hypoxic conditions for an additional 24 hours and luciferase levels were measured using the Luciferase Assay System with Reporter Lysis Buffer kit (Promega).
Article Title: Hypoxia-induced CTCF promotes EMT in breast cancer.
Article Snippet: .. For manipulation of HIF-1a expression in dCa9-TET1 transfected normoxic cells, we transfect these cells with a plasmid constitutively expressing a stable form of HIF-1a (HA-HIF1alpha P402A/P564A-pBabe-puro), which was a gift from William Kaelin (Addgene plasmid # 19005; http://n2t.net/addgene:19005; RRID: Addgene_19005,26). ..
Article Title: Hypoxia-induced CTCF promotes EMT in breast cancer.
Article Snippet: To induce varying degree of CTCF downregulation under hypoxia, different concentration of doxycycline (Sigma, D3072) was added in the cell culture medium and cells were kept in hypoxia. .. For stable HIF-1a expression in normoxic cells, we transfectDNMT3A Knockdown cells with a plasmid constitutively expressing a stable form of HIF-1a (HA-HIF1alpha P402A/P564A-pBabe-puro), which was a gift from William Kaelin (Addgene plasmid # 19005; http://n2t.net/addgene:19005; RRID: Addgene_19005,26). sgRNA designing and cloning in dCas9-DNMT3A or dCas9-TET1 expression vector and cell transfections Guide sequences targeting the loci of interest were selected using the GT-Scan web-tool (http://gt-scan.braembl.org.au/gt-scan/,39) and CRISPick (https://portals.broadinstitute. org/gppx/crispick/public40). .. All sgRNAwere synthesized by oligo annealing and cloned into dCas9-DNMT3A (Addgene #74407) or dCas9-TET1 (Addgene #83340) expression vectors via BbsI (Fermentas, Cat No. ER1012) sites as described previously.25,27 Additionally, non-targeting (NT) control guide RNAs (sgcontrol) was also cloned into dCas9-TET1 (Addgene #83340) expression vector and Addgene #71830 having non-targeting (NT) control guide RNAs (sgcontrol) was used for dCas9-DNMT3A system.
Knockdown:Article Title: Hypoxia-induced CTCF promotes EMT in breast cancer.
Article Snippet: To induce varying degree of CTCF downregulation under hypoxia, different concentration of doxycycline (Sigma, D3072) was added in the cell culture medium and cells were kept in hypoxia. .. For stable HIF-1a expression in normoxic cells, we transfectDNMT3A Knockdown cells with a plasmid constitutively expressing a stable form of HIF-1a (HA-HIF1alpha P402A/P564A-pBabe-puro), which was a gift from William Kaelin (Addgene plasmid # 19005; http://n2t.net/addgene:19005; RRID: Addgene_19005,26). sgRNA designing and cloning in dCas9-DNMT3A or dCas9-TET1 expression vector and cell transfections Guide sequences targeting the loci of interest were selected using the GT-Scan web-tool (http://gt-scan.braembl.org.au/gt-scan/,39) and CRISPick (https://portals.broadinstitute. org/gppx/crispick/public40). .. All sgRNAwere synthesized by oligo annealing and cloned into dCas9-DNMT3A (Addgene #74407) or dCas9-TET1 (Addgene #83340) expression vectors via BbsI (Fermentas, Cat No. ER1012) sites as described previously.25,27 Additionally, non-targeting (NT) control guide RNAs (sgcontrol) was also cloned into dCas9-TET1 (Addgene #83340) expression vector and Addgene #71830 having non-targeting (NT) control guide RNAs (sgcontrol) was used for dCas9-DNMT3A system.
Cloning:Article Title: Hypoxia-induced CTCF promotes EMT in breast cancer.
Article Snippet: To induce varying degree of CTCF downregulation under hypoxia, different concentration of doxycycline (Sigma, D3072) was added in the cell culture medium and cells were kept in hypoxia. .. For stable HIF-1a expression in normoxic cells, we transfectDNMT3A Knockdown cells with a plasmid constitutively expressing a stable form of HIF-1a (HA-HIF1alpha P402A/P564A-pBabe-puro), which was a gift from William Kaelin (Addgene plasmid # 19005; http://n2t.net/addgene:19005; RRID: Addgene_19005,26). sgRNA designing and cloning in dCas9-DNMT3A or dCas9-TET1 expression vector and cell transfections Guide sequences targeting the loci of interest were selected using the GT-Scan web-tool (http://gt-scan.braembl.org.au/gt-scan/,39) and CRISPick (https://portals.broadinstitute. org/gppx/crispick/public40). .. All sgRNAwere synthesized by oligo annealing and cloned into dCas9-DNMT3A (Addgene #74407) or dCas9-TET1 (Addgene #83340) expression vectors via BbsI (Fermentas, Cat No. ER1012) sites as described previously.25,27 Additionally, non-targeting (NT) control guide RNAs (sgcontrol) was also cloned into dCas9-TET1 (Addgene #83340) expression vector and Addgene #71830 having non-targeting (NT) control guide RNAs (sgcontrol) was used for dCas9-DNMT3A system.
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