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Addgene inc pml iv
Pml Iv, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Construct:

Article Title: Transcriptional regulation of telomeric repeat-containing RNA by the G-quadruplex-binding Ewing sarcoma protein.
Article Snippet: We cloned EWS cDNA into pGEX6P-1 (Cytiva, Tokyo, JP, 27-1540-01#) between EcoRI and XhoI to express an N-terminal GST fusion protein (pGEX6P-1-EWS) using EWS forward and reverse primers36,37. pGEX6P-1-RGG3 was constructed by PCR using pGEX6P-1-EWS as a template and contained amino acids 545–656 of EWS cloned into pGEX6P-1 using RGG3 forward and reverse primers. .. Flag-tagged EWS (1–656; Flag-EWS) and EWS lacking the N-terminal region (287–656; Flag-ΔN-term) were constructed by PCR using pGEX6P-1-EWS as the template, pLPC-Flag (Addgene, Massachusetts, USA, 123475#) as the vector using EWS forward and reverse primers or ΔN-term forward and reverse primers, respectively. ..

Article Title: Transcriptional regulation of telomeric repeat-containing RNA by the G-quadruplex-binding Ewing sarcoma protein
Article Snippet: We cloned EWS cDNA into pGEX6P-1 (Cytiva, Tokyo, JP, 27-1540-01#) between Eco RI and Xho I to express an N-terminal GST fusion protein (pGEX6P-1-EWS) using EWS forward and reverse primers , . pGEX6P-1-RGG3 was constructed by PCR using pGEX6P-1-EWS as a template and contained amino acids 545–656 of EWS cloned into pGEX6P-1 using RGG3 forward and reverse primers. .. Flag-tagged EWS (1–656; Flag-EWS) and EWS lacking the N-terminal region (287–656; Flag-ΔN-term) were constructed by PCR using pGEX6P-1-EWS as the template, pLPC-Flag (Addgene, Massachusetts, USA, 123475#) as the vector using EWS forward and reverse primers or ΔN-term forward and reverse primers, respectively. ..

Polymerase Chain Reaction:

Article Title: Transcriptional regulation of telomeric repeat-containing RNA by the G-quadruplex-binding Ewing sarcoma protein.
Article Snippet: We cloned EWS cDNA into pGEX6P-1 (Cytiva, Tokyo, JP, 27-1540-01#) between EcoRI and XhoI to express an N-terminal GST fusion protein (pGEX6P-1-EWS) using EWS forward and reverse primers36,37. pGEX6P-1-RGG3 was constructed by PCR using pGEX6P-1-EWS as a template and contained amino acids 545–656 of EWS cloned into pGEX6P-1 using RGG3 forward and reverse primers. .. Flag-tagged EWS (1–656; Flag-EWS) and EWS lacking the N-terminal region (287–656; Flag-ΔN-term) were constructed by PCR using pGEX6P-1-EWS as the template, pLPC-Flag (Addgene, Massachusetts, USA, 123475#) as the vector using EWS forward and reverse primers or ΔN-term forward and reverse primers, respectively. ..

Article Title: Transcriptional regulation of telomeric repeat-containing RNA by the G-quadruplex-binding Ewing sarcoma protein
Article Snippet: We cloned EWS cDNA into pGEX6P-1 (Cytiva, Tokyo, JP, 27-1540-01#) between Eco RI and Xho I to express an N-terminal GST fusion protein (pGEX6P-1-EWS) using EWS forward and reverse primers , . pGEX6P-1-RGG3 was constructed by PCR using pGEX6P-1-EWS as a template and contained amino acids 545–656 of EWS cloned into pGEX6P-1 using RGG3 forward and reverse primers. .. Flag-tagged EWS (1–656; Flag-EWS) and EWS lacking the N-terminal region (287–656; Flag-ΔN-term) were constructed by PCR using pGEX6P-1-EWS as the template, pLPC-Flag (Addgene, Massachusetts, USA, 123475#) as the vector using EWS forward and reverse primers or ΔN-term forward and reverse primers, respectively. ..

Plasmid Preparation:

Article Title: Transcriptional regulation of telomeric repeat-containing RNA by the G-quadruplex-binding Ewing sarcoma protein.
Article Snippet: We cloned EWS cDNA into pGEX6P-1 (Cytiva, Tokyo, JP, 27-1540-01#) between EcoRI and XhoI to express an N-terminal GST fusion protein (pGEX6P-1-EWS) using EWS forward and reverse primers36,37. pGEX6P-1-RGG3 was constructed by PCR using pGEX6P-1-EWS as a template and contained amino acids 545–656 of EWS cloned into pGEX6P-1 using RGG3 forward and reverse primers. .. Flag-tagged EWS (1–656; Flag-EWS) and EWS lacking the N-terminal region (287–656; Flag-ΔN-term) were constructed by PCR using pGEX6P-1-EWS as the template, pLPC-Flag (Addgene, Massachusetts, USA, 123475#) as the vector using EWS forward and reverse primers or ΔN-term forward and reverse primers, respectively. ..

Article Title: Progerin induces a phenotypic switch in vascular smooth muscle cells and triggers replication stress and an aging-associated secretory signature
Article Snippet: After selection, cells were sorted using EDTA, to ensure that the cells are in a single cell suspension, and filtering using 1/2–1/3 the diameter to sort them using BD FACSCanto II. .. AoSMC-Flag/Flag-progerin: Retroviral particles containing pLPC-Flag and PLPC-Flag-progerin which were a gift from Gerardo Ferbeyre (Addgene plasmid # 69,059 and PLPC-Flag-progerin #69,061) were used to infect AoSMC, and the cells infected were selected with puromycin 4 μγ/ml. .. AoSMC inducible progerin-expressing cells: Lentiviral particles containing pLenti-CMV-TRE3GNeo-GFP progerin, gift from Tom Misteli (Addgene plasmid # 118,710); pLenti-CMV-TRE3G-GFP-laminA, gift from Tom Misteli (Addgene plasmid # 118,709); and the constitutive tetracycline repressor A3 mutant expressing pLenti-CMV-rtTA3, gift from Eric Campau (Addgene plasmid # 26,429) were used to infect AoSMC cells as described in [ 40 ].

Article Title: Transcriptional regulation of telomeric repeat-containing RNA by the G-quadruplex-binding Ewing sarcoma protein
Article Snippet: We cloned EWS cDNA into pGEX6P-1 (Cytiva, Tokyo, JP, 27-1540-01#) between Eco RI and Xho I to express an N-terminal GST fusion protein (pGEX6P-1-EWS) using EWS forward and reverse primers , . pGEX6P-1-RGG3 was constructed by PCR using pGEX6P-1-EWS as a template and contained amino acids 545–656 of EWS cloned into pGEX6P-1 using RGG3 forward and reverse primers. .. Flag-tagged EWS (1–656; Flag-EWS) and EWS lacking the N-terminal region (287–656; Flag-ΔN-term) were constructed by PCR using pGEX6P-1-EWS as the template, pLPC-Flag (Addgene, Massachusetts, USA, 123475#) as the vector using EWS forward and reverse primers or ΔN-term forward and reverse primers, respectively. ..

Retroviral:

Article Title: Progerin induces a phenotypic switch in vascular smooth muscle cells and triggers replication stress and an aging-associated secretory signature
Article Snippet: After selection, cells were sorted using EDTA, to ensure that the cells are in a single cell suspension, and filtering using 1/2–1/3 the diameter to sort them using BD FACSCanto II. .. AoSMC-Flag/Flag-progerin: Retroviral particles containing pLPC-Flag and PLPC-Flag-progerin which were a gift from Gerardo Ferbeyre (Addgene plasmid # 69,059 and PLPC-Flag-progerin #69,061) were used to infect AoSMC, and the cells infected were selected with puromycin 4 μγ/ml. .. AoSMC inducible progerin-expressing cells: Lentiviral particles containing pLenti-CMV-TRE3GNeo-GFP progerin, gift from Tom Misteli (Addgene plasmid # 118,710); pLenti-CMV-TRE3G-GFP-laminA, gift from Tom Misteli (Addgene plasmid # 118,709); and the constitutive tetracycline repressor A3 mutant expressing pLenti-CMV-rtTA3, gift from Eric Campau (Addgene plasmid # 26,429) were used to infect AoSMC cells as described in [ 40 ].

Infection:

Article Title: Progerin induces a phenotypic switch in vascular smooth muscle cells and triggers replication stress and an aging-associated secretory signature
Article Snippet: After selection, cells were sorted using EDTA, to ensure that the cells are in a single cell suspension, and filtering using 1/2–1/3 the diameter to sort them using BD FACSCanto II. .. AoSMC-Flag/Flag-progerin: Retroviral particles containing pLPC-Flag and PLPC-Flag-progerin which were a gift from Gerardo Ferbeyre (Addgene plasmid # 69,059 and PLPC-Flag-progerin #69,061) were used to infect AoSMC, and the cells infected were selected with puromycin 4 μγ/ml. .. AoSMC inducible progerin-expressing cells: Lentiviral particles containing pLenti-CMV-TRE3GNeo-GFP progerin, gift from Tom Misteli (Addgene plasmid # 118,710); pLenti-CMV-TRE3G-GFP-laminA, gift from Tom Misteli (Addgene plasmid # 118,709); and the constitutive tetracycline repressor A3 mutant expressing pLenti-CMV-rtTA3, gift from Eric Campau (Addgene plasmid # 26,429) were used to infect AoSMC cells as described in [ 40 ].



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a , Experimental scheme of genome-wide CRISPR–Cas9 Knockout <t>GecKO</t> <t>v2</t> Library B screening in primary murine macrophages. b , Representative plot of backbone (LGP) or library-B-infected macrophages exposed or not to conditioned media from Pten −/− Trp53 −/− tumor cells. c , Two independent experiments were performed. Graphs show the correlations between the distribution of the guides found in the CD206 − MHCII + population (MHCII) and in the CD206 bright MHCII − population (CD206) from the two experiments. Lib1, library 1; Lib2, library 2. d , Volcano plot showing genes related to the differentially enriched sgRNA guides from CD206 − MHCII + versus CD206 bright MHCII − cells. Negative regulators of the CD206 bright MHCII − population are shown in light blue, and positive regulators are shown in red. log 2 FC ± 0.56, P < 0.005. Statistical analyses and comparisons from NGS output were performed with MAGeCK. e , Western blot analysis showing the percentage of expression of total STAT6. Two independent sgRNA guides (g1 and g2) were utilized to silence Stat6 in macrophages ( n = 2). f , g , FACS analysis of control (LGP) and Stat6 -silenced (g1 and g2) macrophages following exposure to Pten −/− Trp53 −/− conditioned media, with events gated on F4/80 + CD11b + cells: LGP n = 5, g1 n = 5, g2 n = 6 ( f ); LGP n = 4, g1 n = 4, g2 n = 4 ( g ). h , Proliferation of CD8 + T cells exposed to supernatant from Untr. and CM-tr. macrophages: bar graph shows the number of divisions. i , Scratch assay: graph and curves showing the distance (μm) covered by tumor cells over time after exposure to supernatant from Untr. or CM-tr. macrophages ( n = 8). Statistical analyses were performed using two-tailed unpaired Student’s t -test. Values are presented as the mean ± s.e.m. All replicates represent biological replicates. Schematic in a created using BioRender.com .
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a , Experimental scheme of genome-wide CRISPR–Cas9 Knockout GecKO v2 Library B screening in primary murine macrophages. b , Representative plot of backbone (LGP) or library-B-infected macrophages exposed or not to conditioned media from Pten −/− Trp53 −/− tumor cells. c , Two independent experiments were performed. Graphs show the correlations between the distribution of the guides found in the CD206 − MHCII + population (MHCII) and in the CD206 bright MHCII − population (CD206) from the two experiments. Lib1, library 1; Lib2, library 2. d , Volcano plot showing genes related to the differentially enriched sgRNA guides from CD206 − MHCII + versus CD206 bright MHCII − cells. Negative regulators of the CD206 bright MHCII − population are shown in light blue, and positive regulators are shown in red. log 2 FC ± 0.56, P < 0.005. Statistical analyses and comparisons from NGS output were performed with MAGeCK. e , Western blot analysis showing the percentage of expression of total STAT6. Two independent sgRNA guides (g1 and g2) were utilized to silence Stat6 in macrophages ( n = 2). f , g , FACS analysis of control (LGP) and Stat6 -silenced (g1 and g2) macrophages following exposure to Pten −/− Trp53 −/− conditioned media, with events gated on F4/80 + CD11b + cells: LGP n = 5, g1 n = 5, g2 n = 6 ( f ); LGP n = 4, g1 n = 4, g2 n = 4 ( g ). h , Proliferation of CD8 + T cells exposed to supernatant from Untr. and CM-tr. macrophages: bar graph shows the number of divisions. i , Scratch assay: graph and curves showing the distance (μm) covered by tumor cells over time after exposure to supernatant from Untr. or CM-tr. macrophages ( n = 8). Statistical analyses were performed using two-tailed unpaired Student’s t -test. Values are presented as the mean ± s.e.m. All replicates represent biological replicates. Schematic in a created using BioRender.com .

Journal: Nature Immunology

Article Title: Chemosensor receptors are lipid-detecting regulators of macrophage function in cancer

doi: 10.1038/s41590-025-02191-x

Figure Lengend Snippet: a , Experimental scheme of genome-wide CRISPR–Cas9 Knockout GecKO v2 Library B screening in primary murine macrophages. b , Representative plot of backbone (LGP) or library-B-infected macrophages exposed or not to conditioned media from Pten −/− Trp53 −/− tumor cells. c , Two independent experiments were performed. Graphs show the correlations between the distribution of the guides found in the CD206 − MHCII + population (MHCII) and in the CD206 bright MHCII − population (CD206) from the two experiments. Lib1, library 1; Lib2, library 2. d , Volcano plot showing genes related to the differentially enriched sgRNA guides from CD206 − MHCII + versus CD206 bright MHCII − cells. Negative regulators of the CD206 bright MHCII − population are shown in light blue, and positive regulators are shown in red. log 2 FC ± 0.56, P < 0.005. Statistical analyses and comparisons from NGS output were performed with MAGeCK. e , Western blot analysis showing the percentage of expression of total STAT6. Two independent sgRNA guides (g1 and g2) were utilized to silence Stat6 in macrophages ( n = 2). f , g , FACS analysis of control (LGP) and Stat6 -silenced (g1 and g2) macrophages following exposure to Pten −/− Trp53 −/− conditioned media, with events gated on F4/80 + CD11b + cells: LGP n = 5, g1 n = 5, g2 n = 6 ( f ); LGP n = 4, g1 n = 4, g2 n = 4 ( g ). h , Proliferation of CD8 + T cells exposed to supernatant from Untr. and CM-tr. macrophages: bar graph shows the number of divisions. i , Scratch assay: graph and curves showing the distance (μm) covered by tumor cells over time after exposure to supernatant from Untr. or CM-tr. macrophages ( n = 8). Statistical analyses were performed using two-tailed unpaired Student’s t -test. Values are presented as the mean ± s.e.m. All replicates represent biological replicates. Schematic in a created using BioRender.com .

Article Snippet: The mouse GeCKO v2 Library B (Addgene) was used: this library consists of 62,804 sgRNAs constructs, with three sgRNAs targeting each of the 20,661 genes of the mouse genome.

Techniques: Genome Wide, CRISPR, Knock-Out, Infection, Western Blot, Expressing, Control, Wound Healing Assay, Two Tailed Test