Structured Review

OriGene dna sequences
Effect of up- and down-regulation of the expression of <t>DNAJB6</t> on HCMV gene expression, and viral genomic synthesis and production. (A) Western blot analysis of the levels of HCMV IE1, UL44, and UL99 in the parental U251 cells (U251) (lane 1), cells overexpressing DNAJB6a (U251-6a) (lane 4) and b (U251-6b) (lane 6), or U251 cells that were transfected with either anti-DNAJB6a (6a-siRNA) (lane 3) and DNAJB6b siRNA (6b-siRNA) (lane 5) or control siRNA (C-siRNA) (lane 2). The expression of cellular actin was used as the internal control. At 48 hours posttransfection, cells were infected with HCMV (MOI = 1). Protein samples were prepared at 48–72 hours postinfection. (B) To assay the level of intracellular viral <t>DNA,</t> cells were harvested at 72 hours postinfection. To assay the level of viral production, total infection cultures were collected at 5 days postinfection and viral titers were determined. The values of the relative levels of HCMV DNA and titers, which are the means from triplicate experiments, represent the ratios of the levels of viral DNA or titers in different cells to those in the parental U251 cells (U251), respectively. The analyses were repeated three times and the standard deviation is indicated by the error bar.
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Images

1) Product Images from "A Hsp40 Chaperone Protein Interacts with and Modulates the Cellular Distribution of the Primase Protein of Human Cytomegalovirus"

Article Title: A Hsp40 Chaperone Protein Interacts with and Modulates the Cellular Distribution of the Primase Protein of Human Cytomegalovirus

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002968

Effect of up- and down-regulation of the expression of DNAJB6 on HCMV gene expression, and viral genomic synthesis and production. (A) Western blot analysis of the levels of HCMV IE1, UL44, and UL99 in the parental U251 cells (U251) (lane 1), cells overexpressing DNAJB6a (U251-6a) (lane 4) and b (U251-6b) (lane 6), or U251 cells that were transfected with either anti-DNAJB6a (6a-siRNA) (lane 3) and DNAJB6b siRNA (6b-siRNA) (lane 5) or control siRNA (C-siRNA) (lane 2). The expression of cellular actin was used as the internal control. At 48 hours posttransfection, cells were infected with HCMV (MOI = 1). Protein samples were prepared at 48–72 hours postinfection. (B) To assay the level of intracellular viral DNA, cells were harvested at 72 hours postinfection. To assay the level of viral production, total infection cultures were collected at 5 days postinfection and viral titers were determined. The values of the relative levels of HCMV DNA and titers, which are the means from triplicate experiments, represent the ratios of the levels of viral DNA or titers in different cells to those in the parental U251 cells (U251), respectively. The analyses were repeated three times and the standard deviation is indicated by the error bar.
Figure Legend Snippet: Effect of up- and down-regulation of the expression of DNAJB6 on HCMV gene expression, and viral genomic synthesis and production. (A) Western blot analysis of the levels of HCMV IE1, UL44, and UL99 in the parental U251 cells (U251) (lane 1), cells overexpressing DNAJB6a (U251-6a) (lane 4) and b (U251-6b) (lane 6), or U251 cells that were transfected with either anti-DNAJB6a (6a-siRNA) (lane 3) and DNAJB6b siRNA (6b-siRNA) (lane 5) or control siRNA (C-siRNA) (lane 2). The expression of cellular actin was used as the internal control. At 48 hours posttransfection, cells were infected with HCMV (MOI = 1). Protein samples were prepared at 48–72 hours postinfection. (B) To assay the level of intracellular viral DNA, cells were harvested at 72 hours postinfection. To assay the level of viral production, total infection cultures were collected at 5 days postinfection and viral titers were determined. The values of the relative levels of HCMV DNA and titers, which are the means from triplicate experiments, represent the ratios of the levels of viral DNA or titers in different cells to those in the parental U251 cells (U251), respectively. The analyses were repeated three times and the standard deviation is indicated by the error bar.

Techniques Used: Expressing, Western Blot, Transfection, Infection, Standard Deviation

Related Articles

Clone Assay:

Article Title: Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss
Article Snippet: .. Expression Vectors for TS domain, Full length DNMT1 and PCNA The nucleotide sequence encompassing wild type DNMT1 TS domain (aa 306-620) for HeLa cell transfection was custom synthesized (Origene) and this cDNA was cloned into the pCMV6-N-GFP vector. .. The plasmid pEX-N-His-TS containing wild type TS domain (aa 351-600) for bacterial expression and purification was also custom synthesized (Origene).

Transfection:

Article Title: Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells
Article Snippet: .. To generate a stable ALDH1A1 overexpression (ALDH1A1+ ), breast cancer cells were seeded on 6-multiplates and transfected with lentiviral particles containing nucleotide sequences encoding for ALDH1A1 (Origene RC200723 LentiORF particles, ALDH1A1 (Myc-DDK tagged) - Human). .. ALDH1A1+ cells were generated by G418 (400 μg/ml) selection for 10 days.

Article Title: Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss
Article Snippet: .. Expression Vectors for TS domain, Full length DNMT1 and PCNA The nucleotide sequence encompassing wild type DNMT1 TS domain (aa 306-620) for HeLa cell transfection was custom synthesized (Origene) and this cDNA was cloned into the pCMV6-N-GFP vector. .. The plasmid pEX-N-His-TS containing wild type TS domain (aa 351-600) for bacterial expression and purification was also custom synthesized (Origene).

shRNA:

Article Title: Endogenous Glucagon-like Peptide-1 Receptor Signaling in the Nucleus Tractus Solitarius is Required for Food Intake Control
Article Snippet: .. RNA sequences were screened in vitro (OriGene Technologies, Rockland, MD) for their ability to reduce GLP-1R expression according to previously published methods ( ) to create a short hairpin RNA (shRNA) targeting the GLP-1R transcript. .. Briefly, a plasmid designed to express GLP-1Rs ( ; OriGene) was transiently transfected alone or in combination with an shRNA to reduce GLP-1R expression in a rat immortalized hypothalamic neuronal cell line (R-19; Cedarlane Laboratories, Burlington, NC).

In Vitro:

Article Title: Endogenous Glucagon-like Peptide-1 Receptor Signaling in the Nucleus Tractus Solitarius is Required for Food Intake Control
Article Snippet: .. RNA sequences were screened in vitro (OriGene Technologies, Rockland, MD) for their ability to reduce GLP-1R expression according to previously published methods ( ) to create a short hairpin RNA (shRNA) targeting the GLP-1R transcript. .. Briefly, a plasmid designed to express GLP-1Rs ( ; OriGene) was transiently transfected alone or in combination with an shRNA to reduce GLP-1R expression in a rat immortalized hypothalamic neuronal cell line (R-19; Cedarlane Laboratories, Burlington, NC).

Synthesized:

Article Title: Binary architecture of the Nav1.2-β2 signaling complex
Article Snippet: .. Two-electrode voltage-clamp recording from Xenopus oocytes The DNA sequence of hNav 1.2 (NM_021007.2), rNav 1.2a (NM_012647.1), rβ4 (NM_001008880) and hβ2 (NM_004588.4) (acquired from Origene, USA), as well as their mutants was confirmed by automated DNA sequencing and cRNA was synthesized using T7 polymerase (mMessage mMachine kit, Ambion) after linearizing the DNA with appropriate restriction enzymes. .. Channels were expressed in Xenopus oocytes together with a β-subunit (1:5 molar ratio) and studied following 1–2 days incubation after cRNA injection (incubated at 17°C in 96 mM NaCl, 2 mM KCl, 5 mM HEPES, 1 mM MgCl2 and 1.8 mM CaCl2 , 50 μg/ml gentamycin, pH 7.6 with NaOH) using two-electrode voltage-clamp recording techniques (OC-725C, Warner Instruments) with a 150 μl recording chamber.

Article Title: Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss
Article Snippet: .. Expression Vectors for TS domain, Full length DNMT1 and PCNA The nucleotide sequence encompassing wild type DNMT1 TS domain (aa 306-620) for HeLa cell transfection was custom synthesized (Origene) and this cDNA was cloned into the pCMV6-N-GFP vector. .. The plasmid pEX-N-His-TS containing wild type TS domain (aa 351-600) for bacterial expression and purification was also custom synthesized (Origene).

Construct:

Article Title: A Hsp40 Chaperone Protein Interacts with and Modulates the Cellular Distribution of the Primase Protein of Human Cytomegalovirus
Article Snippet: .. The constructs containing the DNA sequences of DNAJB6 isoform a and b were purchased from Origene, Inc. (Rockville, MD). .. To generate constructs pGADT7-DNAJB6b and pRK11-FLAG-DNAJB6b used for expression in yeast and in human cells, the coding sequence of DNAJB6 isoform b (DNAJB6b) was amplified using the primers DNAJB6b-F ( 5′-TTGGCCATGGAGGCCGCGGCCGCGATGGTGGATTACTATGAA-3′ ) and DNAJB6b-R ( 5′-AACGAATTCTGCAGGTTACTTGTTATCCAAGCG-3′ ) and then inserted into SfiI/EcoRI-digested pGADT7 and NotI/PstI-digested pRK11-FLAG, respectively.

Sequencing:

Article Title: Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification
Article Snippet: .. The DNA sequence encoding human DHODH was PCR-amplified from a cDNA template (Origene, SC128197) using Δ29DHODH forward primer: 5′-ACGACAAGCATATGGCCACGGGAGATGAGCG-3′ and Δ29DHODH reverse primer: 5′-GCGACCCGAATTCGGCCGCCGATGATCTGCTCCA ATGGC-3′. .. The PCR reactions contained 1xGC-Rich Buffer (NEB), DMSO (4%), Phusion High-fidelity DNA Polymerase (NEB) (0.05 unit/μL), dNTPs (2 mM), MgCl2 (1.5 mM), cDNA template (20 ng) and primers (1 μM each).

Article Title: Binary architecture of the Nav1.2-β2 signaling complex
Article Snippet: .. Two-electrode voltage-clamp recording from Xenopus oocytes The DNA sequence of hNav 1.2 (NM_021007.2), rNav 1.2a (NM_012647.1), rβ4 (NM_001008880) and hβ2 (NM_004588.4) (acquired from Origene, USA), as well as their mutants was confirmed by automated DNA sequencing and cRNA was synthesized using T7 polymerase (mMessage mMachine kit, Ambion) after linearizing the DNA with appropriate restriction enzymes. .. Channels were expressed in Xenopus oocytes together with a β-subunit (1:5 molar ratio) and studied following 1–2 days incubation after cRNA injection (incubated at 17°C in 96 mM NaCl, 2 mM KCl, 5 mM HEPES, 1 mM MgCl2 and 1.8 mM CaCl2 , 50 μg/ml gentamycin, pH 7.6 with NaOH) using two-electrode voltage-clamp recording techniques (OC-725C, Warner Instruments) with a 150 μl recording chamber.

Article Title: Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss
Article Snippet: .. Expression Vectors for TS domain, Full length DNMT1 and PCNA The nucleotide sequence encompassing wild type DNMT1 TS domain (aa 306-620) for HeLa cell transfection was custom synthesized (Origene) and this cDNA was cloned into the pCMV6-N-GFP vector. .. The plasmid pEX-N-His-TS containing wild type TS domain (aa 351-600) for bacterial expression and purification was also custom synthesized (Origene).

DNA Sequencing:

Article Title: Binary architecture of the Nav1.2-β2 signaling complex
Article Snippet: .. Two-electrode voltage-clamp recording from Xenopus oocytes The DNA sequence of hNav 1.2 (NM_021007.2), rNav 1.2a (NM_012647.1), rβ4 (NM_001008880) and hβ2 (NM_004588.4) (acquired from Origene, USA), as well as their mutants was confirmed by automated DNA sequencing and cRNA was synthesized using T7 polymerase (mMessage mMachine kit, Ambion) after linearizing the DNA with appropriate restriction enzymes. .. Channels were expressed in Xenopus oocytes together with a β-subunit (1:5 molar ratio) and studied following 1–2 days incubation after cRNA injection (incubated at 17°C in 96 mM NaCl, 2 mM KCl, 5 mM HEPES, 1 mM MgCl2 and 1.8 mM CaCl2 , 50 μg/ml gentamycin, pH 7.6 with NaOH) using two-electrode voltage-clamp recording techniques (OC-725C, Warner Instruments) with a 150 μl recording chamber.

Expressing:

Article Title: Endogenous Glucagon-like Peptide-1 Receptor Signaling in the Nucleus Tractus Solitarius is Required for Food Intake Control
Article Snippet: .. RNA sequences were screened in vitro (OriGene Technologies, Rockland, MD) for their ability to reduce GLP-1R expression according to previously published methods ( ) to create a short hairpin RNA (shRNA) targeting the GLP-1R transcript. .. Briefly, a plasmid designed to express GLP-1Rs ( ; OriGene) was transiently transfected alone or in combination with an shRNA to reduce GLP-1R expression in a rat immortalized hypothalamic neuronal cell line (R-19; Cedarlane Laboratories, Burlington, NC).

Article Title: Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss
Article Snippet: .. Expression Vectors for TS domain, Full length DNMT1 and PCNA The nucleotide sequence encompassing wild type DNMT1 TS domain (aa 306-620) for HeLa cell transfection was custom synthesized (Origene) and this cDNA was cloned into the pCMV6-N-GFP vector. .. The plasmid pEX-N-His-TS containing wild type TS domain (aa 351-600) for bacterial expression and purification was also custom synthesized (Origene).

Polymerase Chain Reaction:

Article Title: Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification
Article Snippet: .. The DNA sequence encoding human DHODH was PCR-amplified from a cDNA template (Origene, SC128197) using Δ29DHODH forward primer: 5′-ACGACAAGCATATGGCCACGGGAGATGAGCG-3′ and Δ29DHODH reverse primer: 5′-GCGACCCGAATTCGGCCGCCGATGATCTGCTCCA ATGGC-3′. .. The PCR reactions contained 1xGC-Rich Buffer (NEB), DMSO (4%), Phusion High-fidelity DNA Polymerase (NEB) (0.05 unit/μL), dNTPs (2 mM), MgCl2 (1.5 mM), cDNA template (20 ng) and primers (1 μM each).

Over Expression:

Article Title: Stemness marker ALDH1A1 promotes tumor angiogenesis via retinoic acid/HIF-1α/VEGF signalling in MCF-7 breast cancer cells
Article Snippet: .. To generate a stable ALDH1A1 overexpression (ALDH1A1+ ), breast cancer cells were seeded on 6-multiplates and transfected with lentiviral particles containing nucleotide sequences encoding for ALDH1A1 (Origene RC200723 LentiORF particles, ALDH1A1 (Myc-DDK tagged) - Human). .. ALDH1A1+ cells were generated by G418 (400 μg/ml) selection for 10 days.

Plasmid Preparation:

Article Title: Mutations in DNMT1 cause hereditary sensory neuropathy with dementia and hearing loss
Article Snippet: .. Expression Vectors for TS domain, Full length DNMT1 and PCNA The nucleotide sequence encompassing wild type DNMT1 TS domain (aa 306-620) for HeLa cell transfection was custom synthesized (Origene) and this cDNA was cloned into the pCMV6-N-GFP vector. .. The plasmid pEX-N-His-TS containing wild type TS domain (aa 351-600) for bacterial expression and purification was also custom synthesized (Origene).

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    OriGene nqo1 dna sequence
    3.3. Influence of <t>NQO1</t> on E2 -3,4-Q metabolism and <t>DNA</t> adduct formation
    Nqo1 Dna Sequence, supplied by OriGene, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nqo1 dna sequence/product/OriGene
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    84
    OriGene hairpin sh rna sequence targeting neat1
    <t>NEAT1</t> knockdown impairs GSC tumorigenicity. ( A ) Real time PCR analysis for NEAT1 on GSC#1 and GSC#83 miR-370-3p transduced lines confirmed the down-regulation of the lncRNA. ** p
    Hairpin Sh Rna Sequence Targeting Neat1, supplied by OriGene, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hairpin sh rna sequence targeting neat1/product/OriGene
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    88
    OriGene complementary dna sequences
    Representative standard curve, amplification plot, and dissociation curve, and amplification plot from the patients' samples. (A) NADH dehydrogenase 1 complementary <t>DNA</t> was serially (1∶10) diluted to prepare a series of calibrators (mtDNA standard) with known DNA copy number. The assay was linear over the range (8.48–848000 copies) of DNA copy numbers ( R 2 = 0.997866). (B) The amplification plot (ΔRn versus cycle [log] view) shows no irregular amplification for the standard diluents (848,000, 84,800, 8,480, 848, 84.8, 8.48). (C) The dissociation curve shows a single melting temperature of the specific products generated with standard template. Also, there is no melting temperature observed in the no-template control wells (blank). These dissociation curves indicate that the reactions are free of primer-dimer or any other spurious products. (D) Similar amplification plots were observed after qPCR using patients' samples. The amplification curves generated from human samples were paralleled with the curves from standards and were in the range of amplification plots for standard diluents.
    Complementary Dna Sequences, supplied by OriGene, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complementary dna sequences/product/OriGene
    Average 88 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
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    91
    OriGene short hairpin rna shrna sequences
    Parameters of Ca 2+ homeostasis in YAC128 MSNs with silencing of HAP1A. (A) Immunoblots of HAP1 and beta-actin in YAC128 MSN cultures that overexpressed short-hairpin <t>RNA</t> <t>(shRNA)</t> against HAP1. shRNAs against HAP1 are named a, b, c, d, control scrambled shRNA, and control MSN cultures as indicated on the blot. (B) Protocol to induce SOCE in YAC128 MSNs with silencing of HAP1 using the eight-well system. Cultures of MSNs on DIV14 from YAC128 mice that overexpressed different shRNAs against HAP1 in pLenti-GFP were loaded with the Ca 2+ indicator Fura-2AM and incubated in Ca 2+ -free medium (0.1 mM EGTA). Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (C) Ca 2+ release from the ER in YAC128 MSNs that overexpressed different shRNAs against HAP1. (D) Ca 2+ influx via DHPG-induced SOCE in YAC128 MSNs that overexpressed different shRNAs against HAP1. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. The results of three independent MSN culture preparations are shown. *** p
    Short Hairpin Rna Shrna Sequences, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/short hairpin rna shrna sequences/product/OriGene
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    3.3. Influence of NQO1 on E2 -3,4-Q metabolism and DNA adduct formation

    Journal: The Journal of steroid biochemistry and molecular biology

    Article Title: NAD(P)H:quinone oxidoreductase 1 Arg139Trp and Pro187Ser polymorphisms imbalance estrogen metabolism towards DNA adduct formation in human mammary epithelial cells

    doi: 10.1016/j.jsbmb.2009.07.003

    Figure Lengend Snippet: 3.3. Influence of NQO1 on E2 -3,4-Q metabolism and DNA adduct formation

    Article Snippet: Following validation of the NQO1 DNA sequence (with vector primer v1.5 and vector primer XL39, Origene), this plasmid was used as a template for generating three mutant clones; one contained an AAALys 135 TAAStop mutation in exon 4 of the NQO1 gene (Entrez GeneID: 1728), a second contained the polymorphic CGGArg 139 TGGTrp mutation in exon 4, and a third contained the polymorphic CCTPro 187 TCTSer mutation in exon 6.

    Techniques:

    Analysis of estrogen metabolism and DNA adduct formation in MCF-10F cells expressing wild-type and polymorphic NQO1 variants.

    Journal: The Journal of steroid biochemistry and molecular biology

    Article Title: NAD(P)H:quinone oxidoreductase 1 Arg139Trp and Pro187Ser polymorphisms imbalance estrogen metabolism towards DNA adduct formation in human mammary epithelial cells

    doi: 10.1016/j.jsbmb.2009.07.003

    Figure Lengend Snippet: Analysis of estrogen metabolism and DNA adduct formation in MCF-10F cells expressing wild-type and polymorphic NQO1 variants.

    Article Snippet: Following validation of the NQO1 DNA sequence (with vector primer v1.5 and vector primer XL39, Origene), this plasmid was used as a template for generating three mutant clones; one contained an AAALys 135 TAAStop mutation in exon 4 of the NQO1 gene (Entrez GeneID: 1728), a second contained the polymorphic CGGArg 139 TGGTrp mutation in exon 4, and a third contained the polymorphic CCTPro 187 TCTSer mutation in exon 6.

    Techniques: Expressing

    NEAT1 knockdown impairs GSC tumorigenicity. ( A ) Real time PCR analysis for NEAT1 on GSC#1 and GSC#83 miR-370-3p transduced lines confirmed the down-regulation of the lncRNA. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Mir-370-3p Impairs Glioblastoma Stem-Like Cell Malignancy Regulating a Complex Interplay between HMGA2/HIF1A and the Oncogenic Long Non-Coding RNA (lncRNA) NEAT1

    doi: 10.3390/ijms21103610

    Figure Lengend Snippet: NEAT1 knockdown impairs GSC tumorigenicity. ( A ) Real time PCR analysis for NEAT1 on GSC#1 and GSC#83 miR-370-3p transduced lines confirmed the down-regulation of the lncRNA. ** p

    Article Snippet: The short hairpin (sh)RNA sequence targeting NEAT1 (5′-gatccctaagctgtagaacat-3′, DOI: 10.1002/jcp.27093) was synthesized and cloned in GFP-C-shLenti-vector from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

    Techniques: Real-time Polymerase Chain Reaction

    Schematic drawing of miR-370-3p regulation of HMGA2-HIF1A-NEAT1 interplay and effect on GBM tumorigenesis. Representation of the mechanism proposed in the present study. miR-370-3p down-regulation leads to increased expression of the lncRNA NEAT1, the Epithelial to Mesenchymal Transition (EMT)-inducer HMGA2, the master transcriptional regulator HIF1A, which in turn activates NT5E expression. NEAT1 inhibits the expression of miR-370-3p, promoting the up-regulation of HMGA2 and HIF1A and contributing to GBM progression.

    Journal: International Journal of Molecular Sciences

    Article Title: Mir-370-3p Impairs Glioblastoma Stem-Like Cell Malignancy Regulating a Complex Interplay between HMGA2/HIF1A and the Oncogenic Long Non-Coding RNA (lncRNA) NEAT1

    doi: 10.3390/ijms21103610

    Figure Lengend Snippet: Schematic drawing of miR-370-3p regulation of HMGA2-HIF1A-NEAT1 interplay and effect on GBM tumorigenesis. Representation of the mechanism proposed in the present study. miR-370-3p down-regulation leads to increased expression of the lncRNA NEAT1, the Epithelial to Mesenchymal Transition (EMT)-inducer HMGA2, the master transcriptional regulator HIF1A, which in turn activates NT5E expression. NEAT1 inhibits the expression of miR-370-3p, promoting the up-regulation of HMGA2 and HIF1A and contributing to GBM progression.

    Article Snippet: The short hairpin (sh)RNA sequence targeting NEAT1 (5′-gatccctaagctgtagaacat-3′, DOI: 10.1002/jcp.27093) was synthesized and cloned in GFP-C-shLenti-vector from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

    Techniques: Expressing

    Nuclear Enriched Abundant Transcript 1 (NEAT1) is a target of miR-370-3p and its expression in GBM tissues and GSC lines is inversely correlated with miR-370-3p. ( A ) Binding sites between miR-370-3p and NEAT1 at +1500 (left panel) and at +6500 (right panel) nucleotides of RNA sequence. ( B ) Dual-luciferase reporter assays in 293T cells co-transfected with reporter vectors containing the wild type (wt) or the mutated (mut) NEAT1(1500) (left panel) or NEAT1(6500) (right panel) sequences and miR-370-3p-mimic or control mimic RNA (ctrl). Bar charts show normalized mean values of the relative luciferase activity. Error bars represent the mean ± SD ( n = 4) (student t-test: ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Mir-370-3p Impairs Glioblastoma Stem-Like Cell Malignancy Regulating a Complex Interplay between HMGA2/HIF1A and the Oncogenic Long Non-Coding RNA (lncRNA) NEAT1

    doi: 10.3390/ijms21103610

    Figure Lengend Snippet: Nuclear Enriched Abundant Transcript 1 (NEAT1) is a target of miR-370-3p and its expression in GBM tissues and GSC lines is inversely correlated with miR-370-3p. ( A ) Binding sites between miR-370-3p and NEAT1 at +1500 (left panel) and at +6500 (right panel) nucleotides of RNA sequence. ( B ) Dual-luciferase reporter assays in 293T cells co-transfected with reporter vectors containing the wild type (wt) or the mutated (mut) NEAT1(1500) (left panel) or NEAT1(6500) (right panel) sequences and miR-370-3p-mimic or control mimic RNA (ctrl). Bar charts show normalized mean values of the relative luciferase activity. Error bars represent the mean ± SD ( n = 4) (student t-test: ** p

    Article Snippet: The short hairpin (sh)RNA sequence targeting NEAT1 (5′-gatccctaagctgtagaacat-3′, DOI: 10.1002/jcp.27093) was synthesized and cloned in GFP-C-shLenti-vector from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

    Techniques: Expressing, Binding Assay, Sequencing, Luciferase, Transfection, Activity Assay

    Correlation between miR-370-3p and NEAT1 expression and overall survival (OS) of GBM patients. ( A ) Kaplan–Meier curve for OS in our cohort of GBM patients stratified for low miR-370-3p expression (

    Journal: International Journal of Molecular Sciences

    Article Title: Mir-370-3p Impairs Glioblastoma Stem-Like Cell Malignancy Regulating a Complex Interplay between HMGA2/HIF1A and the Oncogenic Long Non-Coding RNA (lncRNA) NEAT1

    doi: 10.3390/ijms21103610

    Figure Lengend Snippet: Correlation between miR-370-3p and NEAT1 expression and overall survival (OS) of GBM patients. ( A ) Kaplan–Meier curve for OS in our cohort of GBM patients stratified for low miR-370-3p expression (

    Article Snippet: The short hairpin (sh)RNA sequence targeting NEAT1 (5′-gatccctaagctgtagaacat-3′, DOI: 10.1002/jcp.27093) was synthesized and cloned in GFP-C-shLenti-vector from OriGene (OriGene Technologies, Inc., Rockville, MD, USA).

    Techniques: Expressing

    Representative standard curve, amplification plot, and dissociation curve, and amplification plot from the patients' samples. (A) NADH dehydrogenase 1 complementary DNA was serially (1∶10) diluted to prepare a series of calibrators (mtDNA standard) with known DNA copy number. The assay was linear over the range (8.48–848000 copies) of DNA copy numbers ( R 2 = 0.997866). (B) The amplification plot (ΔRn versus cycle [log] view) shows no irregular amplification for the standard diluents (848,000, 84,800, 8,480, 848, 84.8, 8.48). (C) The dissociation curve shows a single melting temperature of the specific products generated with standard template. Also, there is no melting temperature observed in the no-template control wells (blank). These dissociation curves indicate that the reactions are free of primer-dimer or any other spurious products. (D) Similar amplification plots were observed after qPCR using patients' samples. The amplification curves generated from human samples were paralleled with the curves from standards and were in the range of amplification plots for standard diluents.

    Journal: PLoS Medicine

    Article Title: Circulating Mitochondrial DNA in Patients in the ICU as a Marker of Mortality: Derivation and Validation

    doi: 10.1371/journal.pmed.1001577

    Figure Lengend Snippet: Representative standard curve, amplification plot, and dissociation curve, and amplification plot from the patients' samples. (A) NADH dehydrogenase 1 complementary DNA was serially (1∶10) diluted to prepare a series of calibrators (mtDNA standard) with known DNA copy number. The assay was linear over the range (8.48–848000 copies) of DNA copy numbers ( R 2 = 0.997866). (B) The amplification plot (ΔRn versus cycle [log] view) shows no irregular amplification for the standard diluents (848,000, 84,800, 8,480, 848, 84.8, 8.48). (C) The dissociation curve shows a single melting temperature of the specific products generated with standard template. Also, there is no melting temperature observed in the no-template control wells (blank). These dissociation curves indicate that the reactions are free of primer-dimer or any other spurious products. (D) Similar amplification plots were observed after qPCR using patients' samples. The amplification curves generated from human samples were paralleled with the curves from standards and were in the range of amplification plots for standard diluents.

    Article Snippet: Plasmid DNA with complementary DNA sequences for human mtDNA was obtained from ORIGENE (SC101172), and plasmid DNA with complementary DNA sequences for human nuclear DNA was obtained from Sino Biological.

    Techniques: Amplification, Generated, Real-time Polymerase Chain Reaction

    Parameters of Ca 2+ homeostasis in YAC128 MSNs with silencing of HAP1A. (A) Immunoblots of HAP1 and beta-actin in YAC128 MSN cultures that overexpressed short-hairpin RNA (shRNA) against HAP1. shRNAs against HAP1 are named a, b, c, d, control scrambled shRNA, and control MSN cultures as indicated on the blot. (B) Protocol to induce SOCE in YAC128 MSNs with silencing of HAP1 using the eight-well system. Cultures of MSNs on DIV14 from YAC128 mice that overexpressed different shRNAs against HAP1 in pLenti-GFP were loaded with the Ca 2+ indicator Fura-2AM and incubated in Ca 2+ -free medium (0.1 mM EGTA). Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (C) Ca 2+ release from the ER in YAC128 MSNs that overexpressed different shRNAs against HAP1. (D) Ca 2+ influx via DHPG-induced SOCE in YAC128 MSNs that overexpressed different shRNAs against HAP1. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. The results of three independent MSN culture preparations are shown. *** p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

    doi: 10.3389/fncel.2018.00381

    Figure Lengend Snippet: Parameters of Ca 2+ homeostasis in YAC128 MSNs with silencing of HAP1A. (A) Immunoblots of HAP1 and beta-actin in YAC128 MSN cultures that overexpressed short-hairpin RNA (shRNA) against HAP1. shRNAs against HAP1 are named a, b, c, d, control scrambled shRNA, and control MSN cultures as indicated on the blot. (B) Protocol to induce SOCE in YAC128 MSNs with silencing of HAP1 using the eight-well system. Cultures of MSNs on DIV14 from YAC128 mice that overexpressed different shRNAs against HAP1 in pLenti-GFP were loaded with the Ca 2+ indicator Fura-2AM and incubated in Ca 2+ -free medium (0.1 mM EGTA). Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (C) Ca 2+ release from the ER in YAC128 MSNs that overexpressed different shRNAs against HAP1. (D) Ca 2+ influx via DHPG-induced SOCE in YAC128 MSNs that overexpressed different shRNAs against HAP1. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. The results of three independent MSN culture preparations are shown. *** p

    Article Snippet: For HAP1 silencing, commercially available short-hairpin RNA (shRNA) sequences in pLenti-GFP (catalog no. TL500930C, Origene) were used.

    Techniques: Western Blot, shRNA, Mouse Assay, Incubation