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  • 99
    Millipore plasmids expressing cas9
    Plasmids Expressing Cas9, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    BestGene Inc expression plasmids
    Expression Plasmids, supplied by BestGene Inc, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Genecopoeia expression plasmids
    Expression Plasmids, supplied by Genecopoeia, used in various techniques. Bioz Stars score: 92/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc expression plasmids
    Expression Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1262 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    OriGene expression plasmids
    Expression Plasmids, supplied by OriGene, used in various techniques. Bioz Stars score: 92/100, based on 426 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher expression plasmids
    Expression Plasmids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 9504 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega expression plasmids
    Expression Plasmids, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 1344 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa expression plasmids
    Expression Plasmids, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1411 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    SABiosciences expression plasmids
    Expression Plasmids, supplied by SABiosciences, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    OriGene il22ra2i3 expression plasmids
    Il22ra2i3 Expression Plasmids, supplied by OriGene, 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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    85
    TaKaRa tta expressing plasmid
    Tta Expressing Plasmid, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega psvl expression plasmids
    Psvl Expression Plasmids, supplied by Promega, used in various techniques. Bioz Stars score: 80/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Addgene inc cas9 expressing plasmid
    Detection and Quantification of Precision Genome Editing by CRISPR-Mediated HDR, Base Editing, and Prime Editing Using DTECT (A) Schematics of the protocol used to identify genomic changes introduced by CRISPR-dependent HDR, base editing, or prime editing. In HDR experiments (blue), HEK293T cells were transfected with <t>Cas9</t> and an sgRNA targeting a gene of interest with or without donor DNA molecules. In base editing experiments (red), HEK293T cells were transfected with BE3 base editors with either control or base editing sgRNAs. Base editing experiments were also conducted in cells stably expressing FNLS-BE3. In prime editing experiments (gray), HEK293T cells were transfected with PE2 with or without pegRNA. gDNA was then extracted from cell populations and subjected to DTECT using adaptors specific for WT (green) or edited (purple) variants. (B) Identification by DTECT of WT and HDR-edited (R209fs*6) TP53 alleles (top), WT and base-edited (Q223*) FANCD2 alleles (middle), and WT and prime-edited (CTT_ins) HEK3 alleles (bottom). Adaptors specific for the WT (CT, CA, and CG; green) or edited (TT and TA; purple) signatures were used in DTECT experiments. Captured samples were subjected to analytical PCR (left, 21 cycles) or qPCR (right). In the HDR experiment, cells were transfected with Cas9, sgRNA, and an ssODN specific for the TP53 locus with or without the HDR stimulatory factor i53. The ssODN was omitted in control reactions. In the base editing experiment, cells were transfected with BE3 and an sgRNA to induce Q223* in FANCD2. In prime editing experiments, cells were transfected with PE2 and pegRNA to introduce a CTT insertion in the HEK3 locus. (C) Graphical representation of the correlation of DTECT- and NGS-based estimations of the frequency of genetic variants introduced by precision genome editing in human and mouse cells and mouse intestinal organoids (n = 62). Data points in the dashed box (frequency
    Cas9 Expressing Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega pgl3 plasmid expressing
    Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). <t>pGL3</t> + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).
    Pgl3 Plasmid Expressing, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa vsvg expressing plasmid
    Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). <t>pGL3</t> + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).
    Vsvg Expressing Plasmid, supplied by TaKaRa, 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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    92
    Millipore expression pet28a plasmid
    Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). <t>pGL3</t> + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).
    Expression Pet28a Plasmid, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Arbor Vita e6 expression plasmids
    Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). <t>pGL3</t> + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).
    E6 Expression Plasmids, supplied by Arbor Vita, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    OriGene shnrf2 expression plasmids
    Loss of Keap1 contributes to LSCC pathogenesis by activating the Nrf2-ROS pathway (A) In vitro cell proliferation of K/P-LSCC and P-LSCC cells (N=3). (B) In vivo tumor growth of K/P-LSCC and P-LSCC tumors (N=8). (C) In vitro cell invasion of K/P-LSCC and P-LSCC cells (N=3). (D) In vivo lung metastasis of K/P-LSCC and P-LSCC cells (N=6). Microscopic metastatic foci in the lung were counted. ). Gene sets related to stem cells, ROS biology, NFkB and Notch are shown. (F–G) FACS analysis (F) and bar graph (G) of intracellular ROS levels of K/P-LSCC and P-LSCC cells. ROS levels were measured by DCFDA staining. (H) Expression of NFkB target genes in K/P-LSCC and P-LSCC cells assayed by qRT-PCR (N=3). (I) Expression of Nrf2 target genes in K/P-LSCC and P-LSCC cells (N = 3). (J) In vitro cell proliferation of K/P-LSCC and P-LSCC cells transduced with control or <t>shNrf2-lentivirus</t> (N = 3). (K) In vivo growth of K/P-LSCC and P-LSCC tumors with and without shNrf2-lentivirus transduction (N=6). (L) Expression of Notch1 target genes in K/P-LSCC and P-LSCC cells (N=3). All data from (A–D and H–L) are presented as mean ± S.E.M. (* P
    Shnrf2 Expression Plasmids, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa plasmids expressing mtdsred2
    Measures of autophagy and mitophagy in neurons after chronic rotenone. Cortical neurons were transfected with <t>mtDsRed2,</t> treated at DIV7 with rotenone (1 nM) or vehicle control for one or two weeks as described in Methods , then fixed. Immunocytochemistry was performed to stain the autophagic vesicle marker LC3. A, Representative neuronal cell body image (compressed z-stack). B, Percentage of neurons in listed conditions containing greater than 4 autophagic puncta (no significant differences; 5 individual experiments, 64-99 neurons/condition). C, Colocalization of mitochondria with LC3-containing puncta (Pearson’s colocalization coefficients of red and green fluorescence, calculated from each individual z-slice; 3 individual experiments with 5 neurons per condition per experiment; no significant differences).
    Plasmids Expressing Mtdsred2, supplied by TaKaRa, 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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    88
    TaKaRa plasmid expressing egfp
    Measures of autophagy and mitophagy in neurons after chronic rotenone. Cortical neurons were transfected with <t>mtDsRed2,</t> treated at DIV7 with rotenone (1 nM) or vehicle control for one or two weeks as described in Methods , then fixed. Immunocytochemistry was performed to stain the autophagic vesicle marker LC3. A, Representative neuronal cell body image (compressed z-stack). B, Percentage of neurons in listed conditions containing greater than 4 autophagic puncta (no significant differences; 5 individual experiments, 64-99 neurons/condition). C, Colocalization of mitochondria with LC3-containing puncta (Pearson’s colocalization coefficients of red and green fluorescence, calculated from each individual z-slice; 3 individual experiments with 5 neurons per condition per experiment; no significant differences).
    Plasmid Expressing Egfp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc plasmids expressing ulk1
    <t>ULK1</t> modulates 26S proteasome functionality. GFPu-1 cells were transfected with (A) control or ULK1 plasmid for 48 h, and (B) the accumulated GFP fluorescence in ULK1 plasmid-expressing cells was captured with a fluorescent microscope; (C) HEK293 cells were transfected with control or ULK1 plasmid for 48 h and chymotrypsin-like activity was measured; (D) The Ulk1 −/− and WT MEF were transfected with Ub G76V -GFP plasmid for 48 h; (E) Chymotrypsin-like activity was measured in the Ulk1 −/− and WT MEF; (F) HUVECs were transfected with control or ULK1 SiRNA for 48 h and chymotrypsin-like activity was measured. The western blots are representative of three independent experiments. *represents p
    Plasmids Expressing Ulk1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Sino Biological enoph1 expressing plasmid
    <t>ULK1</t> modulates 26S proteasome functionality. GFPu-1 cells were transfected with (A) control or ULK1 plasmid for 48 h, and (B) the accumulated GFP fluorescence in ULK1 plasmid-expressing cells was captured with a fluorescent microscope; (C) HEK293 cells were transfected with control or ULK1 plasmid for 48 h and chymotrypsin-like activity was measured; (D) The Ulk1 −/− and WT MEF were transfected with Ub G76V -GFP plasmid for 48 h; (E) Chymotrypsin-like activity was measured in the Ulk1 −/− and WT MEF; (F) HUVECs were transfected with control or ULK1 SiRNA for 48 h and chymotrypsin-like activity was measured. The western blots are representative of three independent experiments. *represents p
    Enoph1 Expressing Plasmid, supplied by Sino Biological, 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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    88
    Addgene inc sox2 expression plasmids
    Effect of G9a on the transcription and protein expression of <t>Sox2</t> in various cell types. (A) The effect of ectopic expression of G9a on Sox2 and Oct4 protein expression in three breast cancer cell lines (MCF7, ZR-75-1, and MDA-MB-231) and two glioblastoma cell lines (U87-MG and T98-G) was determined by immunoblot analysis. Each cell line was transfected with a G9a-expressing plasmid for 24 h and then harvested for analysis. For quantitative analysis of immunoblotting results, the mean density of the Sox2 band was measured with Multi Gauge V3.0 software, and the band density was divided by the density of β-actin to obtain the normalized band density. (B) Cells were transfected with control or G9a expressing plasmids for 24 h, and the effect of ectopic expression of G9a on the transcription of Sox2 was quantitatively analyzed by real-time RT-PCR. (C) The effect of G9a activity on Sox2 protein stability in mouse ESCs. Mouse ESCs were treated with 0.2 μM BIX-01294 for the indicated times and then harvested for immunoblot analysis. (D) The effect of G9a activity on the transcription of Sox2 in mouse ESCs. Mouse ESCs were treated with 0.2 μM BIX-01294 for the indicated times and then harvested for quantitative analysis of Sox2 transcription by real-time RT-PCR. Abbreviations: G9a, G9a-expressing plasmid; Sox2, Sox2-expressing plasmid; C, control plasmid. *P
    Sox2 Expression Plasmids, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Lonza pmaxgfp expressing plasmid
    Effect of G9a on the transcription and protein expression of <t>Sox2</t> in various cell types. (A) The effect of ectopic expression of G9a on Sox2 and Oct4 protein expression in three breast cancer cell lines (MCF7, ZR-75-1, and MDA-MB-231) and two glioblastoma cell lines (U87-MG and T98-G) was determined by immunoblot analysis. Each cell line was transfected with a G9a-expressing plasmid for 24 h and then harvested for analysis. For quantitative analysis of immunoblotting results, the mean density of the Sox2 band was measured with Multi Gauge V3.0 software, and the band density was divided by the density of β-actin to obtain the normalized band density. (B) Cells were transfected with control or G9a expressing plasmids for 24 h, and the effect of ectopic expression of G9a on the transcription of Sox2 was quantitatively analyzed by real-time RT-PCR. (C) The effect of G9a activity on Sox2 protein stability in mouse ESCs. Mouse ESCs were treated with 0.2 μM BIX-01294 for the indicated times and then harvested for immunoblot analysis. (D) The effect of G9a activity on the transcription of Sox2 in mouse ESCs. Mouse ESCs were treated with 0.2 μM BIX-01294 for the indicated times and then harvested for quantitative analysis of Sox2 transcription by real-time RT-PCR. Abbreviations: G9a, G9a-expressing plasmid; Sox2, Sox2-expressing plasmid; C, control plasmid. *P
    Pmaxgfp Expressing Plasmid, supplied by Lonza, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Addgene inc plasmid expressing egfp
    ( A ) Schematic representation of the <t>dCas9-DNMT3A</t> fusion protein in complex with sgRNA and its target DNA sequence. The sgRNA is bound in a cleft between the recognition lobe (RecI, II and III domains) and the nuclease lobe (HNH, RuvC and PI domains) of Cas9 protein. The C–terminus of Cas9 is located on the PAM–interacting (PI) domain and faces the side where the bound genomic DNA protrudes with its 3′ end relative to the sgRNA sequence. The sgRNA is a synthetic fusion between bacterial crRNA and tracrRNA, with guide sequence and tracrRNA part shown in different colors. The catalytic domain of DNMT3A recruits its partner for dimerization and DNMT3L proteins in vivo (dashed lightened symbols). NLS, nuclear localization signal; GS, Gly 4 Ser peptide linker. ( B ) Domain structure of the dCas9–DNMT3A fusion protein. The nuclease-inactivating mutations D10A and H840A of Streptococcus pyogenes Cas9 are indicated. Deactivated Cas9 was fused to the catalytic domain of the human de novo DNA methyltransferase 3A (DNMT3A CD) using a short Gly 4 Ser peptide (GS). The dCas9–DNMT3A is expressed as a bicistronic mRNA, along with puromycin resistance gene (PuroR, shown) or <t>EGFP</t> gene, thus enabling selection of transfected cells. The PuroR (or EGFP) moiety is separated during translation by action of the T2A self-cleaving peptide. The inactive fusion methyltransferase (dCas9-DNMT3A-ANV) for use as a negative control contains an additional substitution (E155A*) in the active site of DNMT3A. 3x FLAG, epitope tag; NLS, nuclear localization signal.
    Plasmid Expressing Egfp, supplied by Addgene inc, 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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    Detection and Quantification of Precision Genome Editing by CRISPR-Mediated HDR, Base Editing, and Prime Editing Using DTECT (A) Schematics of the protocol used to identify genomic changes introduced by CRISPR-dependent HDR, base editing, or prime editing. In HDR experiments (blue), HEK293T cells were transfected with Cas9 and an sgRNA targeting a gene of interest with or without donor DNA molecules. In base editing experiments (red), HEK293T cells were transfected with BE3 base editors with either control or base editing sgRNAs. Base editing experiments were also conducted in cells stably expressing FNLS-BE3. In prime editing experiments (gray), HEK293T cells were transfected with PE2 with or without pegRNA. gDNA was then extracted from cell populations and subjected to DTECT using adaptors specific for WT (green) or edited (purple) variants. (B) Identification by DTECT of WT and HDR-edited (R209fs*6) TP53 alleles (top), WT and base-edited (Q223*) FANCD2 alleles (middle), and WT and prime-edited (CTT_ins) HEK3 alleles (bottom). Adaptors specific for the WT (CT, CA, and CG; green) or edited (TT and TA; purple) signatures were used in DTECT experiments. Captured samples were subjected to analytical PCR (left, 21 cycles) or qPCR (right). In the HDR experiment, cells were transfected with Cas9, sgRNA, and an ssODN specific for the TP53 locus with or without the HDR stimulatory factor i53. The ssODN was omitted in control reactions. In the base editing experiment, cells were transfected with BE3 and an sgRNA to induce Q223* in FANCD2. In prime editing experiments, cells were transfected with PE2 and pegRNA to introduce a CTT insertion in the HEK3 locus. (C) Graphical representation of the correlation of DTECT- and NGS-based estimations of the frequency of genetic variants introduced by precision genome editing in human and mouse cells and mouse intestinal organoids (n = 62). Data points in the dashed box (frequency

    Journal: Cell reports

    Article Title: Detection of Marker-Free Precision Genome Editing and Genetic Variation through the Capture of Genomic Signatures

    doi: 10.1016/j.celrep.2020.02.068

    Figure Lengend Snippet: Detection and Quantification of Precision Genome Editing by CRISPR-Mediated HDR, Base Editing, and Prime Editing Using DTECT (A) Schematics of the protocol used to identify genomic changes introduced by CRISPR-dependent HDR, base editing, or prime editing. In HDR experiments (blue), HEK293T cells were transfected with Cas9 and an sgRNA targeting a gene of interest with or without donor DNA molecules. In base editing experiments (red), HEK293T cells were transfected with BE3 base editors with either control or base editing sgRNAs. Base editing experiments were also conducted in cells stably expressing FNLS-BE3. In prime editing experiments (gray), HEK293T cells were transfected with PE2 with or without pegRNA. gDNA was then extracted from cell populations and subjected to DTECT using adaptors specific for WT (green) or edited (purple) variants. (B) Identification by DTECT of WT and HDR-edited (R209fs*6) TP53 alleles (top), WT and base-edited (Q223*) FANCD2 alleles (middle), and WT and prime-edited (CTT_ins) HEK3 alleles (bottom). Adaptors specific for the WT (CT, CA, and CG; green) or edited (TT and TA; purple) signatures were used in DTECT experiments. Captured samples were subjected to analytical PCR (left, 21 cycles) or qPCR (right). In the HDR experiment, cells were transfected with Cas9, sgRNA, and an ssODN specific for the TP53 locus with or without the HDR stimulatory factor i53. The ssODN was omitted in control reactions. In the base editing experiment, cells were transfected with BE3 and an sgRNA to induce Q223* in FANCD2. In prime editing experiments, cells were transfected with PE2 and pegRNA to introduce a CTT insertion in the HEK3 locus. (C) Graphical representation of the correlation of DTECT- and NGS-based estimations of the frequency of genetic variants introduced by precision genome editing in human and mouse cells and mouse intestinal organoids (n = 62). Data points in the dashed box (frequency

    Article Snippet: Editing of cell lines, organoids and mice To induce CRISPR-mediated HDR editing, HEK293T cells were seeded at 50%–70% confluency into 24-well plates and reverse transfected with 0.25 μg of sgRNA and 0.25 μg of Cas9 expressing plasmid (Addgene #42230) with or without 0.5 μl of ssODN (40 μM) into 100 μl of DMEM without Fetalgro BGS and antibiotics.

    Techniques: CRISPR, Transfection, Stable Transfection, Expressing, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Introduce, Next-Generation Sequencing

    Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). pGL3 + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).

    Journal: PLoS Genetics

    Article Title: New Small Nuclear RNA Gene-Like Transcriptional Units as Sources of Regulatory Transcripts

    doi: 10.1371/journal.pgen.0030001

    Figure Lengend Snippet: Pol III-Dependent Synthesis of Novel Transcription Units (A) Northern blot analysis of human skin fibroblasts and HeLa cells. Results show two bands: the first (detected at about 300 nucleotides) being the 21A endogenous product and the second (of a very high molecular mass) representing CENP-F mRNA. (B) 21A -specific RT-PCR amplification. As expected for nonpolyadenylated transcripts, an efficient amplification product was obtained only in the random hexamers-primed reactions. (C) Promoter activity transfection assay. A specific luciferase-silencing hairpin is transcribed by six PSE/DSE-dependent promoter elements (11A [ H1 RNA], 14A, 21A, 29A, 38A, 51A). A view of the silencing constructs including the hairpin nucleotide sequence is enclosed. The promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE). pGL3 + pRL, negative control; pSHAG- U6, canonical pol III promoter; No promoter, hairpin without PSE/DSE-dependent promoter thus resulting transcriptionally inactive. (D and E) Promoter-activity transfection assay in presence/absence of 20 μM ML-60218 cell-permeable pol III inhibitor or 10 μg/ml α-amanitin pol II–specific inhibitor. Results are reported as luciferase emission of treated versus untreated samples. (F) Real-time RT-PCR analysis of transcript levels for 21A and 29A transcription units, two known pol III–transcribed genes (7SK and 5S rRNA), and one pol II–transcribed gene (c-Myc) in ML-60218 treated/untreated cells, as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to a ML-60218 unaffected pol II housekeeping gene (GAPDH). (G) Real-time RT-PCR analysis of the RNA level of two known pol II–transcribed (c-Myc and GAPDH) and one pol III–transcribed (7SK) genes in α-amanitin treated/untreated cells as resulting after normalization of each treated sample with its untreated counterpart. All the measures were referred to an α-amanitin unaffected pol III gene (5S rRNA).

    Article Snippet: Oligos used to subclone the novel pol III type III promoters within Not I/HinD III restriction sites (uppercase letters) were the following: 11AFprom Not I: 5′-atgcGCGGCCGCatttgcatgtcgctatgtg-3′ 11ARprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccacgcactcagctcgtg-3′ 14AFprom Not I: 5′-atgcGCGGCCGCaactgatgtatgattatatctt-3′ 14ARprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccattattatctcctttgttctgt-3′ 21AFprom Not I: 5′-atgcGCGGCCGCacagctgtagcagatgct-3′ 21ARprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccaccacacttggtcaactat-3′ 29AFprom Not I: 5′-atgcGCGGCCGCttctcacctaaaggagtc-3′ 29ARprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccttctaatcctcctaagatca-3′ 38AFprom Not I: 5′-atgcGCGGCCGCttcactaagatccagtgc-3′ 38Arprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccgattcatgaacacagaatatt3′ 51AFprom Not I: 5′-atgcGCGGCCGCgttgaacatttaactctgtat-3′ 51Arprom HinD III: 5′-gatcAAGCTTcatcaggtggctcccgctgaattggaatccctcatggcacttggagat-3′ In this analysis, the above constructs were cotransfected with a pGL3 plasmid-expressing (Promega) firefly luciferase as a target to be silenced and with a pRL plasmid expressing (Promega) renilla luciferase to which all the determinations were normalized.

    Techniques: Northern Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Activity Assay, Transfection, Luciferase, Construct, Sequencing, Negative Control, Quantitative RT-PCR

    Loss of Keap1 contributes to LSCC pathogenesis by activating the Nrf2-ROS pathway (A) In vitro cell proliferation of K/P-LSCC and P-LSCC cells (N=3). (B) In vivo tumor growth of K/P-LSCC and P-LSCC tumors (N=8). (C) In vitro cell invasion of K/P-LSCC and P-LSCC cells (N=3). (D) In vivo lung metastasis of K/P-LSCC and P-LSCC cells (N=6). Microscopic metastatic foci in the lung were counted. ). Gene sets related to stem cells, ROS biology, NFkB and Notch are shown. (F–G) FACS analysis (F) and bar graph (G) of intracellular ROS levels of K/P-LSCC and P-LSCC cells. ROS levels were measured by DCFDA staining. (H) Expression of NFkB target genes in K/P-LSCC and P-LSCC cells assayed by qRT-PCR (N=3). (I) Expression of Nrf2 target genes in K/P-LSCC and P-LSCC cells (N = 3). (J) In vitro cell proliferation of K/P-LSCC and P-LSCC cells transduced with control or shNrf2-lentivirus (N = 3). (K) In vivo growth of K/P-LSCC and P-LSCC tumors with and without shNrf2-lentivirus transduction (N=6). (L) Expression of Notch1 target genes in K/P-LSCC and P-LSCC cells (N=3). All data from (A–D and H–L) are presented as mean ± S.E.M. (* P

    Journal: Cancer discovery

    Article Title: Role of KEAP1/NRF2 and TP53 mutations in lung squamous cell carcinoma development and radiotherapy response prediction

    doi: 10.1158/2159-8290.CD-16-0127

    Figure Lengend Snippet: Loss of Keap1 contributes to LSCC pathogenesis by activating the Nrf2-ROS pathway (A) In vitro cell proliferation of K/P-LSCC and P-LSCC cells (N=3). (B) In vivo tumor growth of K/P-LSCC and P-LSCC tumors (N=8). (C) In vitro cell invasion of K/P-LSCC and P-LSCC cells (N=3). (D) In vivo lung metastasis of K/P-LSCC and P-LSCC cells (N=6). Microscopic metastatic foci in the lung were counted. ). Gene sets related to stem cells, ROS biology, NFkB and Notch are shown. (F–G) FACS analysis (F) and bar graph (G) of intracellular ROS levels of K/P-LSCC and P-LSCC cells. ROS levels were measured by DCFDA staining. (H) Expression of NFkB target genes in K/P-LSCC and P-LSCC cells assayed by qRT-PCR (N=3). (I) Expression of Nrf2 target genes in K/P-LSCC and P-LSCC cells (N = 3). (J) In vitro cell proliferation of K/P-LSCC and P-LSCC cells transduced with control or shNrf2-lentivirus (N = 3). (K) In vivo growth of K/P-LSCC and P-LSCC tumors with and without shNrf2-lentivirus transduction (N=6). (L) Expression of Notch1 target genes in K/P-LSCC and P-LSCC cells (N=3). All data from (A–D and H–L) are presented as mean ± S.E.M. (* P

    Article Snippet: shNrf2 expression plasmids were purchased from OriGene (TL515053).

    Techniques: In Vitro, In Vivo, FACS, Staining, Expressing, Quantitative RT-PCR, Transduction

    Measures of autophagy and mitophagy in neurons after chronic rotenone. Cortical neurons were transfected with mtDsRed2, treated at DIV7 with rotenone (1 nM) or vehicle control for one or two weeks as described in Methods , then fixed. Immunocytochemistry was performed to stain the autophagic vesicle marker LC3. A, Representative neuronal cell body image (compressed z-stack). B, Percentage of neurons in listed conditions containing greater than 4 autophagic puncta (no significant differences; 5 individual experiments, 64-99 neurons/condition). C, Colocalization of mitochondria with LC3-containing puncta (Pearson’s colocalization coefficients of red and green fluorescence, calculated from each individual z-slice; 3 individual experiments with 5 neurons per condition per experiment; no significant differences).

    Journal: Neurobiology of disease

    Article Title: Integrating multiple aspects of mitochondrial dynamics in neurons: Age-related differences and dynamic changes in a chronic rotenone model

    doi: 10.1016/j.nbd.2010.09.006

    Figure Lengend Snippet: Measures of autophagy and mitophagy in neurons after chronic rotenone. Cortical neurons were transfected with mtDsRed2, treated at DIV7 with rotenone (1 nM) or vehicle control for one or two weeks as described in Methods , then fixed. Immunocytochemistry was performed to stain the autophagic vesicle marker LC3. A, Representative neuronal cell body image (compressed z-stack). B, Percentage of neurons in listed conditions containing greater than 4 autophagic puncta (no significant differences; 5 individual experiments, 64-99 neurons/condition). C, Colocalization of mitochondria with LC3-containing puncta (Pearson’s colocalization coefficients of red and green fluorescence, calculated from each individual z-slice; 3 individual experiments with 5 neurons per condition per experiment; no significant differences).

    Article Snippet: Cells were transfected with plasmids expressing mtDsRed2 (Clontech), mitochondrially-targeted, photoactivatable GFP containing the mitochondrial targeting sequence from cytochrome c oxidase subunit VIII (PA-mtGFP, provided by R.J. Youle, National Institute of Neurological Disease and Stroke at the National Institutes of Health, Bethesda, MD) along with control vector (pSG5), fission protein Drp1, or dominant-interfering mutant dnDrp1K38A ( ; ) (provided by J.M.

    Techniques: Transfection, Immunocytochemistry, Staining, Marker, Fluorescence

    Mitochondrial density after chronic rotenone exposure. Cortical neurons were treated as described previously, and images taken as described in Methods . A, Example of 3D Metamorph reconstruction of fluorescent mitochondria in neuronal cell body for volume measurements. B, Example of soluble GFP-expressing neurites with mtDsRed2 in distal process. C-D, Mitochondrial density in cell body, proximal neurites, or distal neurites after one or two weeks of rotenone exposure or vehicle control. *Rotenone treatment conditions significantly different than vehicle control. In addition, there are age-related changes, unmarked in graph: control-treated neuronal cell bodies have greater mitochondrial density at the two week time point (DIV21) than at the one week time point (DIV14; p=0.005), but are not statistically significantly different in the other regions at those time points.

    Journal: Neurobiology of disease

    Article Title: Integrating multiple aspects of mitochondrial dynamics in neurons: Age-related differences and dynamic changes in a chronic rotenone model

    doi: 10.1016/j.nbd.2010.09.006

    Figure Lengend Snippet: Mitochondrial density after chronic rotenone exposure. Cortical neurons were treated as described previously, and images taken as described in Methods . A, Example of 3D Metamorph reconstruction of fluorescent mitochondria in neuronal cell body for volume measurements. B, Example of soluble GFP-expressing neurites with mtDsRed2 in distal process. C-D, Mitochondrial density in cell body, proximal neurites, or distal neurites after one or two weeks of rotenone exposure or vehicle control. *Rotenone treatment conditions significantly different than vehicle control. In addition, there are age-related changes, unmarked in graph: control-treated neuronal cell bodies have greater mitochondrial density at the two week time point (DIV21) than at the one week time point (DIV14; p=0.005), but are not statistically significantly different in the other regions at those time points.

    Article Snippet: Cells were transfected with plasmids expressing mtDsRed2 (Clontech), mitochondrially-targeted, photoactivatable GFP containing the mitochondrial targeting sequence from cytochrome c oxidase subunit VIII (PA-mtGFP, provided by R.J. Youle, National Institute of Neurological Disease and Stroke at the National Institutes of Health, Bethesda, MD) along with control vector (pSG5), fission protein Drp1, or dominant-interfering mutant dnDrp1K38A ( ; ) (provided by J.M.

    Techniques: Expressing

    Chronic rotenone alters mitochondrial fission and fusion. Primary rat cortical neurons were transfected with mtDsRed2 and PAmtGFP, then imaged under normal culturing conditions at times indicated (B) or treated beginning at DIV7 for 1or 2 weeks with media containing rotenone at listed concentrations or DMSO vehicle control (C-F) . Live images were recorded for 15 min as described in Methods , and fission and fusion events were quantified blindly as described (n = at least 7 independent experiments, with 3-6 neuronal processes imaged per condition in each experiment. A, Sequential images showing example of mitochondrial fusion (2 nd and 3 rd panel: mitochondrion with mtDsRed2 and photoactivated PAmtGFP (yellow) fuses with neighboring mitochondrion (red) with resulting diffusion of GFP into newly fused side) and fission (4 th ). Gray hashed lines outline neuronal process. Scale bar is 2 μm. B, Probability of fission and fusion events occurring in photoactivated mitochondria over 15 min at DIV7 and DIV28 in culture (*Significantly different from DIV28 neurons, p=0.0173, two-tailed binomial test of differences between proportions). C, Probability of fusion events under conditions as noted. *Control vs 1 nM p = 0.0023 two tailed; **0.1 nM vs 1 nM p

    Journal: Neurobiology of disease

    Article Title: Integrating multiple aspects of mitochondrial dynamics in neurons: Age-related differences and dynamic changes in a chronic rotenone model

    doi: 10.1016/j.nbd.2010.09.006

    Figure Lengend Snippet: Chronic rotenone alters mitochondrial fission and fusion. Primary rat cortical neurons were transfected with mtDsRed2 and PAmtGFP, then imaged under normal culturing conditions at times indicated (B) or treated beginning at DIV7 for 1or 2 weeks with media containing rotenone at listed concentrations or DMSO vehicle control (C-F) . Live images were recorded for 15 min as described in Methods , and fission and fusion events were quantified blindly as described (n = at least 7 independent experiments, with 3-6 neuronal processes imaged per condition in each experiment. A, Sequential images showing example of mitochondrial fusion (2 nd and 3 rd panel: mitochondrion with mtDsRed2 and photoactivated PAmtGFP (yellow) fuses with neighboring mitochondrion (red) with resulting diffusion of GFP into newly fused side) and fission (4 th ). Gray hashed lines outline neuronal process. Scale bar is 2 μm. B, Probability of fission and fusion events occurring in photoactivated mitochondria over 15 min at DIV7 and DIV28 in culture (*Significantly different from DIV28 neurons, p=0.0173, two-tailed binomial test of differences between proportions). C, Probability of fusion events under conditions as noted. *Control vs 1 nM p = 0.0023 two tailed; **0.1 nM vs 1 nM p

    Article Snippet: Cells were transfected with plasmids expressing mtDsRed2 (Clontech), mitochondrially-targeted, photoactivatable GFP containing the mitochondrial targeting sequence from cytochrome c oxidase subunit VIII (PA-mtGFP, provided by R.J. Youle, National Institute of Neurological Disease and Stroke at the National Institutes of Health, Bethesda, MD) along with control vector (pSG5), fission protein Drp1, or dominant-interfering mutant dnDrp1K38A ( ; ) (provided by J.M.

    Techniques: Transfection, Diffusion-based Assay, Two Tailed Test

    ULK1 modulates 26S proteasome functionality. GFPu-1 cells were transfected with (A) control or ULK1 plasmid for 48 h, and (B) the accumulated GFP fluorescence in ULK1 plasmid-expressing cells was captured with a fluorescent microscope; (C) HEK293 cells were transfected with control or ULK1 plasmid for 48 h and chymotrypsin-like activity was measured; (D) The Ulk1 −/− and WT MEF were transfected with Ub G76V -GFP plasmid for 48 h; (E) Chymotrypsin-like activity was measured in the Ulk1 −/− and WT MEF; (F) HUVECs were transfected with control or ULK1 SiRNA for 48 h and chymotrypsin-like activity was measured. The western blots are representative of three independent experiments. *represents p

    Journal: PLoS ONE

    Article Title: Upregulation of Unc-51-Like Kinase 1 by Nitric Oxide Stabilizes SIRT1, Independent of Autophagy

    doi: 10.1371/journal.pone.0116165

    Figure Lengend Snippet: ULK1 modulates 26S proteasome functionality. GFPu-1 cells were transfected with (A) control or ULK1 plasmid for 48 h, and (B) the accumulated GFP fluorescence in ULK1 plasmid-expressing cells was captured with a fluorescent microscope; (C) HEK293 cells were transfected with control or ULK1 plasmid for 48 h and chymotrypsin-like activity was measured; (D) The Ulk1 −/− and WT MEF were transfected with Ub G76V -GFP plasmid for 48 h; (E) Chymotrypsin-like activity was measured in the Ulk1 −/− and WT MEF; (F) HUVECs were transfected with control or ULK1 SiRNA for 48 h and chymotrypsin-like activity was measured. The western blots are representative of three independent experiments. *represents p

    Article Snippet: Plasmids expressing ULK1 (Addgene plasmid 31961) were prepared and kindly provided by Dr. Do-Hyung Kim of the University of Minnesota.

    Techniques: Transfection, Plasmid Preparation, Fluorescence, Expressing, Microscopy, Activity Assay, Western Blot

    The NO-ULK1-SIRT1 pathway might be operative in the whole animal. (A) Western blots of lung tissues of wild-type (C57BL/6J) and eNOS knock-out mice; (B) Western blots of lung tissues of wild-type (C57BL/6J) and db/db mice; (C) Western blots of lung tissues of wild-type (C57BL/6J) and db/db mice; (D) The proposed mechanism of the eNOS-derived NO regulation of SIRT1 protein turnover, which requires ULK1 and OGT, in vascular endothelial cells. *represents p

    Journal: PLoS ONE

    Article Title: Upregulation of Unc-51-Like Kinase 1 by Nitric Oxide Stabilizes SIRT1, Independent of Autophagy

    doi: 10.1371/journal.pone.0116165

    Figure Lengend Snippet: The NO-ULK1-SIRT1 pathway might be operative in the whole animal. (A) Western blots of lung tissues of wild-type (C57BL/6J) and eNOS knock-out mice; (B) Western blots of lung tissues of wild-type (C57BL/6J) and db/db mice; (C) Western blots of lung tissues of wild-type (C57BL/6J) and db/db mice; (D) The proposed mechanism of the eNOS-derived NO regulation of SIRT1 protein turnover, which requires ULK1 and OGT, in vascular endothelial cells. *represents p

    Article Snippet: Plasmids expressing ULK1 (Addgene plasmid 31961) were prepared and kindly provided by Dr. Do-Hyung Kim of the University of Minnesota.

    Techniques: Western Blot, Knock-Out, Mouse Assay, Derivative Assay

    ULK1 regulates SIRT1 protein expression via 26S proteasomes. (A) HUVECs were transfected with control or ULK1 SiRNA for 48 h, then treated with epoxomicin (0.1 µM) for 4 h; (B) HUVECs were transfected with control or ULK1 siRNA for 48 h, SIRT1 mRNA levels were determined by RT-PCR; (C) HUVECs were transfected with control or ULK1 or β-TrCP1 siRNA for 48 h. The western blots are representative of three independent experiments. *represents p

    Journal: PLoS ONE

    Article Title: Upregulation of Unc-51-Like Kinase 1 by Nitric Oxide Stabilizes SIRT1, Independent of Autophagy

    doi: 10.1371/journal.pone.0116165

    Figure Lengend Snippet: ULK1 regulates SIRT1 protein expression via 26S proteasomes. (A) HUVECs were transfected with control or ULK1 SiRNA for 48 h, then treated with epoxomicin (0.1 µM) for 4 h; (B) HUVECs were transfected with control or ULK1 siRNA for 48 h, SIRT1 mRNA levels were determined by RT-PCR; (C) HUVECs were transfected with control or ULK1 or β-TrCP1 siRNA for 48 h. The western blots are representative of three independent experiments. *represents p

    Article Snippet: Plasmids expressing ULK1 (Addgene plasmid 31961) were prepared and kindly provided by Dr. Do-Hyung Kim of the University of Minnesota.

    Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot

    ULK1 regulates 26S proteasome functionality via OGT. (A–B) HEK293 cells were transfected with control or ULK1 plasmid for 48 h; (C–D) HUVECs were transfected with control or ULK1 siRNA for 48 h, then treated with NONOate (50 µM) for 4 h. The western blots are representative of three independent experiments. *represents p

    Journal: PLoS ONE

    Article Title: Upregulation of Unc-51-Like Kinase 1 by Nitric Oxide Stabilizes SIRT1, Independent of Autophagy

    doi: 10.1371/journal.pone.0116165

    Figure Lengend Snippet: ULK1 regulates 26S proteasome functionality via OGT. (A–B) HEK293 cells were transfected with control or ULK1 plasmid for 48 h; (C–D) HUVECs were transfected with control or ULK1 siRNA for 48 h, then treated with NONOate (50 µM) for 4 h. The western blots are representative of three independent experiments. *represents p

    Article Snippet: Plasmids expressing ULK1 (Addgene plasmid 31961) were prepared and kindly provided by Dr. Do-Hyung Kim of the University of Minnesota.

    Techniques: Transfection, Plasmid Preparation, Western Blot

    ULK1 regulates SIRT1 protein expression which is independent of autophagy. (A) HEK293 cells were transfected with pcDNA or ULK1 plasmid for 48 h; (B) HEK293 cells were transfected with pcDNA or ULK1 plasmid for 48 h. SIRT1 mRNA levels were determined by RT-PCR; (C) HUVECs were treated with rapamycin (2 µM) for the indicated time; (D) U20S cells which expressed GFP-LC3 reporter were treated with rapamycin (2 µM) or NONOate (50 µM) for 12 h; GFP-LC3 imaging was captured using fluorescent microscope according to instructions of the manufacturer, EMD Millipore; (E) MEF from the Ulk1 −/− , Ulk2 −/− , Ulk1/2 −/− , and WT mice were treated with bafilomycin A1 (10 nM) for 4 h; (F) HUVECs were treated with NONOate (50 µM) for the indicated time; (G) HUVECs were transfected with GFP or eNOS adenovirus for 48 h; (H) HUVECs were treated with A23187 (1 µM) for the indicated time. The western blots are representative of three independent experiments. *represent p

    Journal: PLoS ONE

    Article Title: Upregulation of Unc-51-Like Kinase 1 by Nitric Oxide Stabilizes SIRT1, Independent of Autophagy

    doi: 10.1371/journal.pone.0116165

    Figure Lengend Snippet: ULK1 regulates SIRT1 protein expression which is independent of autophagy. (A) HEK293 cells were transfected with pcDNA or ULK1 plasmid for 48 h; (B) HEK293 cells were transfected with pcDNA or ULK1 plasmid for 48 h. SIRT1 mRNA levels were determined by RT-PCR; (C) HUVECs were treated with rapamycin (2 µM) for the indicated time; (D) U20S cells which expressed GFP-LC3 reporter were treated with rapamycin (2 µM) or NONOate (50 µM) for 12 h; GFP-LC3 imaging was captured using fluorescent microscope according to instructions of the manufacturer, EMD Millipore; (E) MEF from the Ulk1 −/− , Ulk2 −/− , Ulk1/2 −/− , and WT mice were treated with bafilomycin A1 (10 nM) for 4 h; (F) HUVECs were treated with NONOate (50 µM) for the indicated time; (G) HUVECs were transfected with GFP or eNOS adenovirus for 48 h; (H) HUVECs were treated with A23187 (1 µM) for the indicated time. The western blots are representative of three independent experiments. *represent p

    Article Snippet: Plasmids expressing ULK1 (Addgene plasmid 31961) were prepared and kindly provided by Dr. Do-Hyung Kim of the University of Minnesota.

    Techniques: Expressing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Imaging, Microscopy, Mouse Assay, Western Blot

    NO stabilizes and upregulates ULK1 protein expression. (A) HUVECs were transfected with GFP or eNOS adenovirus for 48 h; (B) HUVECs were treated with A23187 (1 µM) for the indicated time; (C) HUVECs were treated with NONOate (50 µM) for the indicated time; (D) HUVECs were treated with CHX (5 µM) for the indicated time, followed by incubation of NONOate (50 µM) for 4 h; (E) HUVECs were treated with CHX (5 µM) for the indicated time, followed by incubation of A23187 (1 µM) for 4 h. The western blots are representative of three independent experiments. *represents p

    Journal: PLoS ONE

    Article Title: Upregulation of Unc-51-Like Kinase 1 by Nitric Oxide Stabilizes SIRT1, Independent of Autophagy

    doi: 10.1371/journal.pone.0116165

    Figure Lengend Snippet: NO stabilizes and upregulates ULK1 protein expression. (A) HUVECs were transfected with GFP or eNOS adenovirus for 48 h; (B) HUVECs were treated with A23187 (1 µM) for the indicated time; (C) HUVECs were treated with NONOate (50 µM) for the indicated time; (D) HUVECs were treated with CHX (5 µM) for the indicated time, followed by incubation of NONOate (50 µM) for 4 h; (E) HUVECs were treated with CHX (5 µM) for the indicated time, followed by incubation of A23187 (1 µM) for 4 h. The western blots are representative of three independent experiments. *represents p

    Article Snippet: Plasmids expressing ULK1 (Addgene plasmid 31961) were prepared and kindly provided by Dr. Do-Hyung Kim of the University of Minnesota.

    Techniques: Expressing, Transfection, Incubation, Western Blot

    ULK1 mediates the NO-induced SIRT1 upregulation. (A) MEF from the Ulk1 −/− , Ulk2 −/− , Ulk1/2 −/− , and WT mice were transfected with GFP or eNOS Adenovirus for 48 h; (B) MEF from the Ulk1 −/− , Ulk2 −/− , Ulk1/2 −/− , and WT mice were treated with NONOate (50 µM) or A23187 (1 µM) for 4 h; (C) HUVECs were transfected with control or ULK1 SiRNA for 48 h, then treated with A23187 (1 µM) for 4 h; (D) HUVECs were transfected with control or ULK1 SiRNA for 48 h, then treated with NONOate (50 µM) for 4 h. The western blots shown are representative of three independent experiments. *represents p

    Journal: PLoS ONE

    Article Title: Upregulation of Unc-51-Like Kinase 1 by Nitric Oxide Stabilizes SIRT1, Independent of Autophagy

    doi: 10.1371/journal.pone.0116165

    Figure Lengend Snippet: ULK1 mediates the NO-induced SIRT1 upregulation. (A) MEF from the Ulk1 −/− , Ulk2 −/− , Ulk1/2 −/− , and WT mice were transfected with GFP or eNOS Adenovirus for 48 h; (B) MEF from the Ulk1 −/− , Ulk2 −/− , Ulk1/2 −/− , and WT mice were treated with NONOate (50 µM) or A23187 (1 µM) for 4 h; (C) HUVECs were transfected with control or ULK1 SiRNA for 48 h, then treated with A23187 (1 µM) for 4 h; (D) HUVECs were transfected with control or ULK1 SiRNA for 48 h, then treated with NONOate (50 µM) for 4 h. The western blots shown are representative of three independent experiments. *represents p

    Article Snippet: Plasmids expressing ULK1 (Addgene plasmid 31961) were prepared and kindly provided by Dr. Do-Hyung Kim of the University of Minnesota.

    Techniques: Mouse Assay, Transfection, Western Blot

    Effect of G9a on the transcription and protein expression of Sox2 in various cell types. (A) The effect of ectopic expression of G9a on Sox2 and Oct4 protein expression in three breast cancer cell lines (MCF7, ZR-75-1, and MDA-MB-231) and two glioblastoma cell lines (U87-MG and T98-G) was determined by immunoblot analysis. Each cell line was transfected with a G9a-expressing plasmid for 24 h and then harvested for analysis. For quantitative analysis of immunoblotting results, the mean density of the Sox2 band was measured with Multi Gauge V3.0 software, and the band density was divided by the density of β-actin to obtain the normalized band density. (B) Cells were transfected with control or G9a expressing plasmids for 24 h, and the effect of ectopic expression of G9a on the transcription of Sox2 was quantitatively analyzed by real-time RT-PCR. (C) The effect of G9a activity on Sox2 protein stability in mouse ESCs. Mouse ESCs were treated with 0.2 μM BIX-01294 for the indicated times and then harvested for immunoblot analysis. (D) The effect of G9a activity on the transcription of Sox2 in mouse ESCs. Mouse ESCs were treated with 0.2 μM BIX-01294 for the indicated times and then harvested for quantitative analysis of Sox2 transcription by real-time RT-PCR. Abbreviations: G9a, G9a-expressing plasmid; Sox2, Sox2-expressing plasmid; C, control plasmid. *P

    Journal: PLoS ONE

    Article Title: Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability

    doi: 10.1371/journal.pone.0141118

    Figure Lengend Snippet: Effect of G9a on the transcription and protein expression of Sox2 in various cell types. (A) The effect of ectopic expression of G9a on Sox2 and Oct4 protein expression in three breast cancer cell lines (MCF7, ZR-75-1, and MDA-MB-231) and two glioblastoma cell lines (U87-MG and T98-G) was determined by immunoblot analysis. Each cell line was transfected with a G9a-expressing plasmid for 24 h and then harvested for analysis. For quantitative analysis of immunoblotting results, the mean density of the Sox2 band was measured with Multi Gauge V3.0 software, and the band density was divided by the density of β-actin to obtain the normalized band density. (B) Cells were transfected with control or G9a expressing plasmids for 24 h, and the effect of ectopic expression of G9a on the transcription of Sox2 was quantitatively analyzed by real-time RT-PCR. (C) The effect of G9a activity on Sox2 protein stability in mouse ESCs. Mouse ESCs were treated with 0.2 μM BIX-01294 for the indicated times and then harvested for immunoblot analysis. (D) The effect of G9a activity on the transcription of Sox2 in mouse ESCs. Mouse ESCs were treated with 0.2 μM BIX-01294 for the indicated times and then harvested for quantitative analysis of Sox2 transcription by real-time RT-PCR. Abbreviations: G9a, G9a-expressing plasmid; Sox2, Sox2-expressing plasmid; C, control plasmid. *P

    Article Snippet: Plasmid, siRNA, and transfection Human G9a, Sox2 expression plasmids were purchased from Addgene (Cambridge, MA) and human G9a siRNAs (L-006937-00-0010) and Sox2 siRNAs (L-011778-00-0005) were obtained from Dharmacon, Inc (Chicago, IL).

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Software, Quantitative RT-PCR, Activity Assay

    Chemical inhibition of G9a activity reduces the stability of Sox2 protein in MCF7 cells. (A) MCF7 cells were treated with the indicated doses of BIX-01294 for 24 h. Whole cell lysates were harvested and analyzed by immunoblotting with G9a-, Sox2-, and Oct4-specific antibodies. (B) MCF7 cells were treated with 0.2 μM of BIX-01294 for 96 h, and whole cell lysates were analyzed for the expression of Sox2 and Oct4. (C) MCF7 cells were treated with 2.0 μM BIX-01294 for 96 h, and the levels of Sox2 transcripts were analyzed by real-time RT-PCR analysis. mRNA levels were normalized to those of GAPDH. (D) Sox2 and Oct4 protein levels in MCF7 cells were analyzed by immunoblotting after cells were treated with 2.0 μM BIX-01294 in the presence of the protein synthesis inhibitor, cycloheximide (5 μM). The graph shows band intensity data normalized according to the controls and β-actin. (E) Cell growth was analyzed by counting cell numbers at the indicated times after treatment with 2.0 μM BIX-01294. For quantitative analysis of the immunoblotting results, the mean density of the Sox2 band was measured with Multi Gauge V3.0 software, and the band density was divided by the density of β-actin to obtain the normalized band density. * P

    Journal: PLoS ONE

    Article Title: Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability

    doi: 10.1371/journal.pone.0141118

    Figure Lengend Snippet: Chemical inhibition of G9a activity reduces the stability of Sox2 protein in MCF7 cells. (A) MCF7 cells were treated with the indicated doses of BIX-01294 for 24 h. Whole cell lysates were harvested and analyzed by immunoblotting with G9a-, Sox2-, and Oct4-specific antibodies. (B) MCF7 cells were treated with 0.2 μM of BIX-01294 for 96 h, and whole cell lysates were analyzed for the expression of Sox2 and Oct4. (C) MCF7 cells were treated with 2.0 μM BIX-01294 for 96 h, and the levels of Sox2 transcripts were analyzed by real-time RT-PCR analysis. mRNA levels were normalized to those of GAPDH. (D) Sox2 and Oct4 protein levels in MCF7 cells were analyzed by immunoblotting after cells were treated with 2.0 μM BIX-01294 in the presence of the protein synthesis inhibitor, cycloheximide (5 μM). The graph shows band intensity data normalized according to the controls and β-actin. (E) Cell growth was analyzed by counting cell numbers at the indicated times after treatment with 2.0 μM BIX-01294. For quantitative analysis of the immunoblotting results, the mean density of the Sox2 band was measured with Multi Gauge V3.0 software, and the band density was divided by the density of β-actin to obtain the normalized band density. * P

    Article Snippet: Plasmid, siRNA, and transfection Human G9a, Sox2 expression plasmids were purchased from Addgene (Cambridge, MA) and human G9a siRNAs (L-006937-00-0010) and Sox2 siRNAs (L-011778-00-0005) were obtained from Dharmacon, Inc (Chicago, IL).

    Techniques: Inhibition, Activity Assay, Expressing, Quantitative RT-PCR, Software

    The expression level of G9a affects the amount of Sox2 protein in MCF7 cells. (A) MCF7 cells were transfected with siNS or siG9a for 24 h, and G9a transcript levels were analyzed by real-time PCR by priming two positions of G9a ORF (G9aI and G9aII). mRNA levels were normalized to those of GAPDH. G9aI and G9a2 primer sets were designed to amplify 1299~1531 bp and 2326~2637 bp of G9a ORF, respectively. (B) The effect of G9a on the proliferation of MCF7 cells. MCF7 cells (1 × 10 4 ) were transfected with siNS or siG9a and then counted at the indicated times post-transfection. (C) MCF-7 cells were transfected with siNS or siG9a for 24 h, and the effect of G9a knockdown on the transcription of Oct4 and Sox2 was quantitatively analyzed by real-time RT-PCR. (D) MCF-7 cells were transfected with siNS or siG9a for 24 h, and the effect of G9a knockdown on the amount of Oct4 and Sox2 protein was analyzed by immunoblot analysis. (E) MCF-7 cells were transfected with control or G9a expression plasmids for 24 h, and the effect of G9a overexpression on the transcription of Sox2 and Oct4 was quantitatively analyzed by real-time RT-PCR. (F) MCF-7 cells were transfected with control or G9a expression plasmids for 24 h, and the effect of G9a overexpression on the amount of Sox2 and Oct4 was quantitatively analyzed by immunoblot analysis. (G) Co-immunoprecipitation of endogenous Sox2 and G9a in MCF7 cells. Total protein extract of MCF7 cells was immunoprecipitated with Sox2 and then immunoblotted with G9a antibody. The reciprocal experiment was also performed in which total protein of MCF7 cells was immunoprecipitated with G9a and then immunoblotted with Sox2 antibody. The specific bands corresponding to the G9a and Sox2 was arrowed. Abbreviations: siNS, non-specific siRNA; siG9a, siRNA targeting G9a; C, control plasmid; OE, G9a-expressing plasmid; IP, immunoprecipitation; IB: immunoblotting. For quantitative analysis of the immunoblotting results, the mean density of the Sox2 or G9a bands was measured with Multi Gauge V3.0 software. The band density was then divided by the density of β-actin to obtain the normalized band density. *P

    Journal: PLoS ONE

    Article Title: Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability

    doi: 10.1371/journal.pone.0141118

    Figure Lengend Snippet: The expression level of G9a affects the amount of Sox2 protein in MCF7 cells. (A) MCF7 cells were transfected with siNS or siG9a for 24 h, and G9a transcript levels were analyzed by real-time PCR by priming two positions of G9a ORF (G9aI and G9aII). mRNA levels were normalized to those of GAPDH. G9aI and G9a2 primer sets were designed to amplify 1299~1531 bp and 2326~2637 bp of G9a ORF, respectively. (B) The effect of G9a on the proliferation of MCF7 cells. MCF7 cells (1 × 10 4 ) were transfected with siNS or siG9a and then counted at the indicated times post-transfection. (C) MCF-7 cells were transfected with siNS or siG9a for 24 h, and the effect of G9a knockdown on the transcription of Oct4 and Sox2 was quantitatively analyzed by real-time RT-PCR. (D) MCF-7 cells were transfected with siNS or siG9a for 24 h, and the effect of G9a knockdown on the amount of Oct4 and Sox2 protein was analyzed by immunoblot analysis. (E) MCF-7 cells were transfected with control or G9a expression plasmids for 24 h, and the effect of G9a overexpression on the transcription of Sox2 and Oct4 was quantitatively analyzed by real-time RT-PCR. (F) MCF-7 cells were transfected with control or G9a expression plasmids for 24 h, and the effect of G9a overexpression on the amount of Sox2 and Oct4 was quantitatively analyzed by immunoblot analysis. (G) Co-immunoprecipitation of endogenous Sox2 and G9a in MCF7 cells. Total protein extract of MCF7 cells was immunoprecipitated with Sox2 and then immunoblotted with G9a antibody. The reciprocal experiment was also performed in which total protein of MCF7 cells was immunoprecipitated with G9a and then immunoblotted with Sox2 antibody. The specific bands corresponding to the G9a and Sox2 was arrowed. Abbreviations: siNS, non-specific siRNA; siG9a, siRNA targeting G9a; C, control plasmid; OE, G9a-expressing plasmid; IP, immunoprecipitation; IB: immunoblotting. For quantitative analysis of the immunoblotting results, the mean density of the Sox2 or G9a bands was measured with Multi Gauge V3.0 software. The band density was then divided by the density of β-actin to obtain the normalized band density. *P

    Article Snippet: Plasmid, siRNA, and transfection Human G9a, Sox2 expression plasmids were purchased from Addgene (Cambridge, MA) and human G9a siRNAs (L-006937-00-0010) and Sox2 siRNAs (L-011778-00-0005) were obtained from Dharmacon, Inc (Chicago, IL).

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression, Immunoprecipitation, Plasmid Preparation, Software

    ( A ) Schematic representation of the dCas9-DNMT3A fusion protein in complex with sgRNA and its target DNA sequence. The sgRNA is bound in a cleft between the recognition lobe (RecI, II and III domains) and the nuclease lobe (HNH, RuvC and PI domains) of Cas9 protein. The C–terminus of Cas9 is located on the PAM–interacting (PI) domain and faces the side where the bound genomic DNA protrudes with its 3′ end relative to the sgRNA sequence. The sgRNA is a synthetic fusion between bacterial crRNA and tracrRNA, with guide sequence and tracrRNA part shown in different colors. The catalytic domain of DNMT3A recruits its partner for dimerization and DNMT3L proteins in vivo (dashed lightened symbols). NLS, nuclear localization signal; GS, Gly 4 Ser peptide linker. ( B ) Domain structure of the dCas9–DNMT3A fusion protein. The nuclease-inactivating mutations D10A and H840A of Streptococcus pyogenes Cas9 are indicated. Deactivated Cas9 was fused to the catalytic domain of the human de novo DNA methyltransferase 3A (DNMT3A CD) using a short Gly 4 Ser peptide (GS). The dCas9–DNMT3A is expressed as a bicistronic mRNA, along with puromycin resistance gene (PuroR, shown) or EGFP gene, thus enabling selection of transfected cells. The PuroR (or EGFP) moiety is separated during translation by action of the T2A self-cleaving peptide. The inactive fusion methyltransferase (dCas9-DNMT3A-ANV) for use as a negative control contains an additional substitution (E155A*) in the active site of DNMT3A. 3x FLAG, epitope tag; NLS, nuclear localization signal.

    Journal: Nucleic Acids Research

    Article Title: Repurposing the CRISPR-Cas9 system for targeted DNA methylation

    doi: 10.1093/nar/gkw159

    Figure Lengend Snippet: ( A ) Schematic representation of the dCas9-DNMT3A fusion protein in complex with sgRNA and its target DNA sequence. The sgRNA is bound in a cleft between the recognition lobe (RecI, II and III domains) and the nuclease lobe (HNH, RuvC and PI domains) of Cas9 protein. The C–terminus of Cas9 is located on the PAM–interacting (PI) domain and faces the side where the bound genomic DNA protrudes with its 3′ end relative to the sgRNA sequence. The sgRNA is a synthetic fusion between bacterial crRNA and tracrRNA, with guide sequence and tracrRNA part shown in different colors. The catalytic domain of DNMT3A recruits its partner for dimerization and DNMT3L proteins in vivo (dashed lightened symbols). NLS, nuclear localization signal; GS, Gly 4 Ser peptide linker. ( B ) Domain structure of the dCas9–DNMT3A fusion protein. The nuclease-inactivating mutations D10A and H840A of Streptococcus pyogenes Cas9 are indicated. Deactivated Cas9 was fused to the catalytic domain of the human de novo DNA methyltransferase 3A (DNMT3A CD) using a short Gly 4 Ser peptide (GS). The dCas9–DNMT3A is expressed as a bicistronic mRNA, along with puromycin resistance gene (PuroR, shown) or EGFP gene, thus enabling selection of transfected cells. The PuroR (or EGFP) moiety is separated during translation by action of the T2A self-cleaving peptide. The inactive fusion methyltransferase (dCas9-DNMT3A-ANV) for use as a negative control contains an additional substitution (E155A*) in the active site of DNMT3A. 3x FLAG, epitope tag; NLS, nuclear localization signal.

    Article Snippet: Western blot analysis of total protein extracts HEK293 cells were co–transfected with 100 ng of plasmid expressing BACH2-sgRNA8 constructs with puromycin selection marker (wild-type dCas9-DNMT3A or ANV-mutant) and 20 ng of plasmid expressing EGFP (pcDNA3-EGFP, Addgene plasmid # 13031, a gift from Doug Golenbock).

    Techniques: Sequencing, In Vivo, Selection, Transfection, Negative Control, FLAG-tag

    ( A ) Time-course evaluation of targeted CpG methylation in cells transfected with active or inactive dCas9-DNMT3A construct co-expressed with individual targeting sgRNAs. CpG methylation level increase was measured at different time points after transfection over a time period of 40 days. We analyzed CpG sites falling within the peak of methylation activity 18–42 bp downstream from the PAM sequence (positions of CpG sites relative to the PAM sequence are given in brackets). The time point when DNA methylation reaches its maximum is marked with a purple dashed vertical line. Left panel: Methylation of the BACH2-A fragment in cells co-expressing BACH2-sgRNA8. Right panel: Methylation of the IL6ST-A fragment in cells co-expressing IL6ST-sgRNA3. ( B ) Relative amount of plasmid DNA per cell, as determined by qPCR, decreases sharply in the 10 days following transfection. ( C ) Even without puromycin selection (using the construct co–expressing EGFP instead of puromycin resistance gene), the detected expression of the Cas9-DNMT3A construct (measured via co-expression of EGFP) decreases within 10 days after transfection. Arbitrary units (AU) represent fluorescence intensity per field of view normalized to the total number of cells counted under visible light.

    Journal: Nucleic Acids Research

    Article Title: Repurposing the CRISPR-Cas9 system for targeted DNA methylation

    doi: 10.1093/nar/gkw159

    Figure Lengend Snippet: ( A ) Time-course evaluation of targeted CpG methylation in cells transfected with active or inactive dCas9-DNMT3A construct co-expressed with individual targeting sgRNAs. CpG methylation level increase was measured at different time points after transfection over a time period of 40 days. We analyzed CpG sites falling within the peak of methylation activity 18–42 bp downstream from the PAM sequence (positions of CpG sites relative to the PAM sequence are given in brackets). The time point when DNA methylation reaches its maximum is marked with a purple dashed vertical line. Left panel: Methylation of the BACH2-A fragment in cells co-expressing BACH2-sgRNA8. Right panel: Methylation of the IL6ST-A fragment in cells co-expressing IL6ST-sgRNA3. ( B ) Relative amount of plasmid DNA per cell, as determined by qPCR, decreases sharply in the 10 days following transfection. ( C ) Even without puromycin selection (using the construct co–expressing EGFP instead of puromycin resistance gene), the detected expression of the Cas9-DNMT3A construct (measured via co-expression of EGFP) decreases within 10 days after transfection. Arbitrary units (AU) represent fluorescence intensity per field of view normalized to the total number of cells counted under visible light.

    Article Snippet: Western blot analysis of total protein extracts HEK293 cells were co–transfected with 100 ng of plasmid expressing BACH2-sgRNA8 constructs with puromycin selection marker (wild-type dCas9-DNMT3A or ANV-mutant) and 20 ng of plasmid expressing EGFP (pcDNA3-EGFP, Addgene plasmid # 13031, a gift from Doug Golenbock).

    Techniques: CpG Methylation Assay, Transfection, Construct, Methylation, Activity Assay, Sequencing, DNA Methylation Assay, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Selection, Fluorescence