gfp vector Search Results


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  • 98
    ATCC lentiviral vector
    <t>Lentiviral</t> (LV)-wild-type (WT)-HSCT corrects behavior. The open-field behavior test was performed for one hour at the same point of the circadian rhythm at 6 months (24 weeks) of age ( n = 10 female mice per group). The measures of hyperactivity were:
    Lentiviral Vector, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 246 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 98 stars, based on 246 article reviews
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    91
    OriGene pcmv6 ac gfp vector
    Effects of LGALS4 protein expression (gal-4) in T24 transfectants The cells were transfected with either the <t>pCMV6-AC-GFP/gal-4</t> (T24/gal-4 cell line) or empty vector (T24/mock cell line) as a control. (A) Ectopic expression of gal-4 in T24 transfectants. Proteins from the whole extracts of T24/mock or T24/gal-4 cells were analyzed by Western analysis for the detection of gal-4 or β-actin (as a loading control). The lane beside the markers was run with the extracts of T24 cells as a contrast. T24/gal-4* was T24 transfectants containing the pIRES-EGFP/gal-4 vector, which showed gal-4 expression (36 kD) only as a contrast. (B) Cell proliferation of T24/mock and T24/gal-4 cells, as determined in cell viability assays using trypan blue exclusion method. Bars represent the mean± SEM of three independent experiments performed in duplicate. ** P
    Pcmv6 Ac Gfp Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    GenePharma Company pgpu6 gfp neo vector
    Aldolase A (ALDOA) knockdown decreased the proliferation capacity of non-small cell lung cancer (NSCLC). a Comparison of the endogenous ALDOA expression of several NSCLC cell lines. All cell lines except H157 displayed abundant expression of ALDOA. Data are shown as mean ± standard deviation (SD). b H520 cells were stably transfected with <t>pGPU6/GFP/Neo-ALDOA-short</t> hairpin RNA (shAL-1, shAL-2) or pGPU6/GFP/Neo-shNC (shNC, negative control). ALDOA was lower in shAL H520 cells than shNC cells. Data are shown as mean ± SD. * P
    Pgpu6 Gfp Neo Vector, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 89/100, based on 314 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 89 stars, based on 314 article reviews
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    90
    Addgene inc pspcas9 bb 2a gfp vector
    Aldolase A (ALDOA) knockdown decreased the proliferation capacity of non-small cell lung cancer (NSCLC). a Comparison of the endogenous ALDOA expression of several NSCLC cell lines. All cell lines except H157 displayed abundant expression of ALDOA. Data are shown as mean ± standard deviation (SD). b H520 cells were stably transfected with <t>pGPU6/GFP/Neo-ALDOA-short</t> hairpin RNA (shAL-1, shAL-2) or pGPU6/GFP/Neo-shNC (shNC, negative control). ALDOA was lower in shAL H520 cells than shNC cells. Data are shown as mean ± SD. * P
    Pspcas9 Bb 2a Gfp Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 306 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Genechem pgcsil gfp vector
    Overexpression of miR-675-5p inhibits NSCLC in vivo. (A) Tumor volumes of subcutaneous implantation models of NSCLC are shown. (B) Tumor growth curves of subcutaneous implantation models of NSCLC. (C) Tumor volumes in the orthotopic implantation models at week 4 are shown. Data were represented as the mean ± SEM of three independent experiments. Negative control: <t>pGCsil-GFP</t> Vector *P
    Pgcsil Gfp Vector, supplied by Genechem, used in various techniques. Bioz Stars score: 91/100, based on 381 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Shanghai Genechem pgcsil gfp vector
    Overexpression of miR-675-5p inhibits NSCLC in vivo. (A) Tumor volumes of subcutaneous implantation models of NSCLC are shown. (B) Tumor growth curves of subcutaneous implantation models of NSCLC. (C) Tumor volumes in the orthotopic implantation models at week 4 are shown. Data were represented as the mean ± SEM of three independent experiments. Negative control: <t>pGCsil-GFP</t> Vector *P
    Pgcsil Gfp Vector, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 89/100, based on 381 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Addgene inc pcag gfp vector
    Studies of the minimum Speedy A fragment that is required for its localization to telomeres. The different regions of Speedy A cDNA were cloned into the <t>pCAG-GFP</t> vector and expressed by electroporation in wild-type testes. ( A ) Western blot showing that
    Pcag Gfp Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    OriGene gfp vector
    <t>GMFG</t> overexpression enhances chemoattractant-stimulated cell migration and adhesion to FN. Human monocytes or THP-1 cells were transfected with <t>GFP</t> vector or GFP-tagged GMFG plasmid for 48 h. A , Western blotting analysis of expression of GMFG or GMFG-GFP
    Gfp Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 91/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    OriGene lentiviral gfp vector
    Downregulation of the α2δ1 subunit impairs the presynaptic release of glutamate and abolishes α2δ1 overexpression-driven enhancement of spontaneous neuronal firing. A , B , shRNA-induced knockdown of the α2δ1 subunit results in significant decrease of its surface expression and in corresponding decrease of the live HA fluorescence in rat hippocampal neurons. C , Western blot demonstrates a significant decrease in neuronal expression of the α2δ1 subunit on shRNA-triggered knockdown. D , A timeline of infection (green triangle) and electrophysiological recordings (orange triangles) shown in E–H . E , Downregulation of the α2δ1 subunit, but not the <t>GFP</t> expression, leads to significant reduction of the mean frequency of mEPSCs in rat hippocampal neurons. F , Cumulative distribution of interevent intervals for mEPSCs recorded under control conditions or on α2δ1 knockdown. G , The mean mEPSC amplitude is not affected by either α2δ1 knockdown, or by <t>lentiviral</t> expression of the GFP. H , Cumulative distribution of mEPSC amplitudes recorded under control conditions or on α2δ1 knockdown. I , A timeline of infection (green triangle) and multichannel recordings (orange triangles) shown in J , K . J , Representative traces of spontaneous neuronal firing in rat hippocampal cultures under control conditions (black), as well as after 1 week of either α2δ1 overexpression (red) or knockdown (brown). Thirty of 60 channels from each array are shown. Scale bar, 10 s. K , The shRNA-mediated knockdown of the α2δ1 subunit during the fourth week in vitro is associated with suppression of the spontaneous neuronal firing. * p
    Lentiviral Gfp Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Addgene inc gfp vector
    TRPV1-WT but not the <t>LWI</t> mutants co-localize with endogenous lipid raft markers. <t>TRPV1-WT-GFP</t> (green) and different LWI mutants in GFP (green) were expressed in F11 cells. Cells were fixed 36 hours post transfection and stained for lipid raft with Cholera Toxin B-594 (red). All images were acquired by confocal microscope. Same experiment was performed with cells that were treated with 5mM MβCD for cholesterol depletion 30 min before cell fixation. TRPV1-WT-GFP shows distinct co-localization with Cholera Toxin B in the membranous region while LWI mutants are distinctly excluded from Cholera Toxin B-enriched membranous regions.
    Gfp Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GenePharma Company lv3 cmv gfp puro vector
    TRPV1-WT but not the <t>LWI</t> mutants co-localize with endogenous lipid raft markers. <t>TRPV1-WT-GFP</t> (green) and different LWI mutants in GFP (green) were expressed in F11 cells. Cells were fixed 36 hours post transfection and stained for lipid raft with Cholera Toxin B-594 (red). All images were acquired by confocal microscope. Same experiment was performed with cells that were treated with 5mM MβCD for cholesterol depletion 30 min before cell fixation. TRPV1-WT-GFP shows distinct co-localization with Cholera Toxin B in the membranous region while LWI mutants are distinctly excluded from Cholera Toxin B-enriched membranous regions.
    Lv3 Cmv Gfp Puro Vector, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 88/100, based on 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GenePharma Company pglv3 h1 gfp puro vector
    TRPV1-WT but not the <t>LWI</t> mutants co-localize with endogenous lipid raft markers. <t>TRPV1-WT-GFP</t> (green) and different LWI mutants in GFP (green) were expressed in F11 cells. Cells were fixed 36 hours post transfection and stained for lipid raft with Cholera Toxin B-594 (red). All images were acquired by confocal microscope. Same experiment was performed with cells that were treated with 5mM MβCD for cholesterol depletion 30 min before cell fixation. TRPV1-WT-GFP shows distinct co-localization with Cholera Toxin B in the membranous region while LWI mutants are distinctly excluded from Cholera Toxin B-enriched membranous regions.
    Pglv3 H1 Gfp Puro Vector, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 88/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Addgene inc gfp expression vector ppd95 77
    TRPV1-WT but not the <t>LWI</t> mutants co-localize with endogenous lipid raft markers. <t>TRPV1-WT-GFP</t> (green) and different LWI mutants in GFP (green) were expressed in F11 cells. Cells were fixed 36 hours post transfection and stained for lipid raft with Cholera Toxin B-594 (red). All images were acquired by confocal microscope. Same experiment was performed with cells that were treated with 5mM MβCD for cholesterol depletion 30 min before cell fixation. TRPV1-WT-GFP shows distinct co-localization with Cholera Toxin B in the membranous region while LWI mutants are distinctly excluded from Cholera Toxin B-enriched membranous regions.
    Gfp Expression Vector Ppd95 77, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher gfp expression vector
    Immunophenotypic characterization of human MSC and MSC-mediated increase in invasiveness of pancreatic cancer cells. A. FACS analysis of human MSC using anti-human CD34, CD45, CD73, and CD105. Mouse IgG as classmatch control for mouse monoclonal CD34, CD45, rabbit IgG for rabbit monoclonal CD73 and CD105. Indirect immunofluorescence using Alexa488-conjugated secondary antibodies. B and C. <t>BxPC3</t> invasiveness was assessed under the co-culture condition with hMSCs by employing Boyden chamber method. Filter membrane set in the top chamber was sealed with Matrigel to close the micro pore. The mixed cells <t>(BxPC3-GFP</t> cells and hMSCs, ratio 1:1) were placed in the top chamber filled with serum free medium and serum was added or not added to the bottom well to be final concentrations 0.5% and 10%. After incubation for 12 hours, passed BxPC3-GFP cells through the filter membrane were observed by the fluorescence microscope (A) and their fluorescence intensities were measured and quantified (B). Data from A are means ± SD, *P
    Gfp Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp  (TaKaRa)
    96
    TaKaRa gfp
    Homing of <t>MSCs</t> in pancreas. (A) Primary culture of MSCs showing many spindle-shaped stem cells (white arrows) ×200. (B) Fluorescent microscopic image of a section in pancreas of rat in group III demonstrating the green fluorescence of MSCs labeled with <t>GFP</t> two week after implantation (white arrows) ×200. (C) Flow cytometric chart analysis for surface antigens of MSCs. They were positive for CD29 and CD90.
    Gfp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 11520 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    GenePharma Company pgpu6 gfp neo shrna expression vector
    Flk-1 <t>shRNA</t> in U87 cells inhibits tumor growth and VM. A, Flk-1 gene knockdown results in suppression of tumor growth. U87 cells expressing Flk-1 <t>shRNA-GFP</t> or GFP vector were injected subcutaneously into SCID/Beige mice, and tumor size was measured weekly
    Pgpu6 Gfp Neo Shrna Expression Vector, supplied by GenePharma Company, used in various techniques. Bioz Stars score: 85/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    OriGene retroviral gfp vector
    Flk-1 <t>shRNA</t> in U87 cells inhibits tumor growth and VM. A, Flk-1 gene knockdown results in suppression of tumor growth. U87 cells expressing Flk-1 <t>shRNA-GFP</t> or GFP vector were injected subcutaneously into SCID/Beige mice, and tumor size was measured weekly
    Retroviral Gfp Vector, supplied by OriGene, used in various techniques. Bioz Stars score: 88/100, based on 77 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Becton Dickinson gfp expression vector
    EBNA3C blocks p73 and Apaf-1 expressions by inhibiting the DNA-binding ability of E2F1 to its targeted promoters in LCLs. A–B) Approximately10 million human peripheral blood mononuclear cells (PBMC) were infected by either A) wild-type (WT) BAC <t>GFP-EBV</t> or EBNA3C knockout BAC GFP-EBV (ΔE3C) for 4 h. At indicates times post-infection cells were harvested, total RNA was isolated and subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1 and EBNA3C transcript levels. Each sample was tested in triplicate and data obtained from two independent experiments with two different donors and expressed as the difference of the quantity of specific transcripts to the quantity of GAPDH transcript as control. The fold change in expression of each mRNA relative to GAPDH transcript was calculated based on the threshold cycle (Ct) as 2 − Δ(ΔCt) , where ΔCt = Ct target −Ct GAPDH and Δ(ΔCt) = ΔCt test sample −ΔCt control sample . C) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins were subjected to western blot (WB) analysis using indicated antibodies. D) Approximately 20 million of EBNA3C knockdown LCLs were <t>transfected</t> with 50 ìg of plasmids expressing either vector control or myc-tagged EBNA3C via electroporation. Transfected LCLs were cultured in RPMI medium with 10% FBS for 48 h and subjected for western blot analysis using indicated antibodies. E) Total RNA was isolated from indicated LCLs, subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1, p73 and Apaf-1 transcript levels as similar to A–B). F) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins (50 µg) were subjected to western blot analysis using indicated antibodies. G) A ChIP assay was performed using either control or EBNA3C knockdown LCLs. Material immunoprecipitated with anti-E2F1 or control antibody (rabbit IgG) was amplified by using primers specific for p73 (top) or Apaf1 (bottom) promoters. The end products of qPCR bands ran into a 2.5% agarose gel. Bar diagrams represent the change in Ct value (ΔCt) over IgG. H–I) Saos-2 (p53 −/− pRb −/− ) cells were transfected with either 5 µg of the wild-type H) p73-luciferase or I) Apaf-1-luciferase reporter plasmids with flag-E2F1 and myc-EBNA3C expression vectors as indicated. Luciferase activity was assessed at 36 h of post-transfection. J) Schematic representation of streptavidin pulldown assay as described in ‘ Materials and Methods ’ section. K) 100 µg of cell extracts from Saos-2 cells transfected with flag-E2F1 with or without myc-EBNA3C expression vector were incubated with 200 ng of the indicated biotinylated oligonucleotides (WT or Mut) immobilized with streptavidin accordingly to the manufacturer protocol, in the absence or presence of a 200 molar excess of the corresponding non biotinylated oligonucleotide (competition: comp). Oligonucleotide-bound E2F1 protein was detected by western blotting using anti-flag antibody (M2). The binding capacity of each oligonucleotide is given as percentage at bottom. All panels are representative of two independent experiments.
    Gfp Expression Vector, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 60 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 90 stars, based on 60 article reviews
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    85
    Addgene inc plenti cmv gfp puro vector
    EBNA3C blocks p73 and Apaf-1 expressions by inhibiting the DNA-binding ability of E2F1 to its targeted promoters in LCLs. A–B) Approximately10 million human peripheral blood mononuclear cells (PBMC) were infected by either A) wild-type (WT) BAC <t>GFP-EBV</t> or EBNA3C knockout BAC GFP-EBV (ΔE3C) for 4 h. At indicates times post-infection cells were harvested, total RNA was isolated and subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1 and EBNA3C transcript levels. Each sample was tested in triplicate and data obtained from two independent experiments with two different donors and expressed as the difference of the quantity of specific transcripts to the quantity of GAPDH transcript as control. The fold change in expression of each mRNA relative to GAPDH transcript was calculated based on the threshold cycle (Ct) as 2 − Δ(ΔCt) , where ΔCt = Ct target −Ct GAPDH and Δ(ΔCt) = ΔCt test sample −ΔCt control sample . C) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins were subjected to western blot (WB) analysis using indicated antibodies. D) Approximately 20 million of EBNA3C knockdown LCLs were <t>transfected</t> with 50 ìg of plasmids expressing either vector control or myc-tagged EBNA3C via electroporation. Transfected LCLs were cultured in RPMI medium with 10% FBS for 48 h and subjected for western blot analysis using indicated antibodies. E) Total RNA was isolated from indicated LCLs, subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1, p73 and Apaf-1 transcript levels as similar to A–B). F) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins (50 µg) were subjected to western blot analysis using indicated antibodies. G) A ChIP assay was performed using either control or EBNA3C knockdown LCLs. Material immunoprecipitated with anti-E2F1 or control antibody (rabbit IgG) was amplified by using primers specific for p73 (top) or Apaf1 (bottom) promoters. The end products of qPCR bands ran into a 2.5% agarose gel. Bar diagrams represent the change in Ct value (ΔCt) over IgG. H–I) Saos-2 (p53 −/− pRb −/− ) cells were transfected with either 5 µg of the wild-type H) p73-luciferase or I) Apaf-1-luciferase reporter plasmids with flag-E2F1 and myc-EBNA3C expression vectors as indicated. Luciferase activity was assessed at 36 h of post-transfection. J) Schematic representation of streptavidin pulldown assay as described in ‘ Materials and Methods ’ section. K) 100 µg of cell extracts from Saos-2 cells transfected with flag-E2F1 with or without myc-EBNA3C expression vector were incubated with 200 ng of the indicated biotinylated oligonucleotides (WT or Mut) immobilized with streptavidin accordingly to the manufacturer protocol, in the absence or presence of a 200 molar excess of the corresponding non biotinylated oligonucleotide (competition: comp). Oligonucleotide-bound E2F1 protein was detected by western blotting using anti-flag antibody (M2). The binding capacity of each oligonucleotide is given as percentage at bottom. All panels are representative of two independent experiments.
    Plenti Cmv Gfp Puro Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM gfp vector pqbi polii
    EBNA3C blocks p73 and Apaf-1 expressions by inhibiting the DNA-binding ability of E2F1 to its targeted promoters in LCLs. A–B) Approximately10 million human peripheral blood mononuclear cells (PBMC) were infected by either A) wild-type (WT) BAC <t>GFP-EBV</t> or EBNA3C knockout BAC GFP-EBV (ΔE3C) for 4 h. At indicates times post-infection cells were harvested, total RNA was isolated and subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1 and EBNA3C transcript levels. Each sample was tested in triplicate and data obtained from two independent experiments with two different donors and expressed as the difference of the quantity of specific transcripts to the quantity of GAPDH transcript as control. The fold change in expression of each mRNA relative to GAPDH transcript was calculated based on the threshold cycle (Ct) as 2 − Δ(ΔCt) , where ΔCt = Ct target −Ct GAPDH and Δ(ΔCt) = ΔCt test sample −ΔCt control sample . C) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins were subjected to western blot (WB) analysis using indicated antibodies. D) Approximately 20 million of EBNA3C knockdown LCLs were <t>transfected</t> with 50 ìg of plasmids expressing either vector control or myc-tagged EBNA3C via electroporation. Transfected LCLs were cultured in RPMI medium with 10% FBS for 48 h and subjected for western blot analysis using indicated antibodies. E) Total RNA was isolated from indicated LCLs, subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1, p73 and Apaf-1 transcript levels as similar to A–B). F) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins (50 µg) were subjected to western blot analysis using indicated antibodies. G) A ChIP assay was performed using either control or EBNA3C knockdown LCLs. Material immunoprecipitated with anti-E2F1 or control antibody (rabbit IgG) was amplified by using primers specific for p73 (top) or Apaf1 (bottom) promoters. The end products of qPCR bands ran into a 2.5% agarose gel. Bar diagrams represent the change in Ct value (ΔCt) over IgG. H–I) Saos-2 (p53 −/− pRb −/− ) cells were transfected with either 5 µg of the wild-type H) p73-luciferase or I) Apaf-1-luciferase reporter plasmids with flag-E2F1 and myc-EBNA3C expression vectors as indicated. Luciferase activity was assessed at 36 h of post-transfection. J) Schematic representation of streptavidin pulldown assay as described in ‘ Materials and Methods ’ section. K) 100 µg of cell extracts from Saos-2 cells transfected with flag-E2F1 with or without myc-EBNA3C expression vector were incubated with 200 ng of the indicated biotinylated oligonucleotides (WT or Mut) immobilized with streptavidin accordingly to the manufacturer protocol, in the absence or presence of a 200 molar excess of the corresponding non biotinylated oligonucleotide (competition: comp). Oligonucleotide-bound E2F1 protein was detected by western blotting using anti-flag antibody (M2). The binding capacity of each oligonucleotide is given as percentage at bottom. All panels are representative of two independent experiments.
    Gfp Vector Pqbi Polii, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    gfp vector pqbi polii - by Bioz Stars, 2020-09
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    90
    Thermo Fisher gfp topo vector
    EBNA3C blocks p73 and Apaf-1 expressions by inhibiting the DNA-binding ability of E2F1 to its targeted promoters in LCLs. A–B) Approximately10 million human peripheral blood mononuclear cells (PBMC) were infected by either A) wild-type (WT) BAC <t>GFP-EBV</t> or EBNA3C knockout BAC GFP-EBV (ΔE3C) for 4 h. At indicates times post-infection cells were harvested, total RNA was isolated and subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1 and EBNA3C transcript levels. Each sample was tested in triplicate and data obtained from two independent experiments with two different donors and expressed as the difference of the quantity of specific transcripts to the quantity of GAPDH transcript as control. The fold change in expression of each mRNA relative to GAPDH transcript was calculated based on the threshold cycle (Ct) as 2 − Δ(ΔCt) , where ΔCt = Ct target −Ct GAPDH and Δ(ΔCt) = ΔCt test sample −ΔCt control sample . C) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins were subjected to western blot (WB) analysis using indicated antibodies. D) Approximately 20 million of EBNA3C knockdown LCLs were <t>transfected</t> with 50 ìg of plasmids expressing either vector control or myc-tagged EBNA3C via electroporation. Transfected LCLs were cultured in RPMI medium with 10% FBS for 48 h and subjected for western blot analysis using indicated antibodies. E) Total RNA was isolated from indicated LCLs, subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1, p73 and Apaf-1 transcript levels as similar to A–B). F) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins (50 µg) were subjected to western blot analysis using indicated antibodies. G) A ChIP assay was performed using either control or EBNA3C knockdown LCLs. Material immunoprecipitated with anti-E2F1 or control antibody (rabbit IgG) was amplified by using primers specific for p73 (top) or Apaf1 (bottom) promoters. The end products of qPCR bands ran into a 2.5% agarose gel. Bar diagrams represent the change in Ct value (ΔCt) over IgG. H–I) Saos-2 (p53 −/− pRb −/− ) cells were transfected with either 5 µg of the wild-type H) p73-luciferase or I) Apaf-1-luciferase reporter plasmids with flag-E2F1 and myc-EBNA3C expression vectors as indicated. Luciferase activity was assessed at 36 h of post-transfection. J) Schematic representation of streptavidin pulldown assay as described in ‘ Materials and Methods ’ section. K) 100 µg of cell extracts from Saos-2 cells transfected with flag-E2F1 with or without myc-EBNA3C expression vector were incubated with 200 ng of the indicated biotinylated oligonucleotides (WT or Mut) immobilized with streptavidin accordingly to the manufacturer protocol, in the absence or presence of a 200 molar excess of the corresponding non biotinylated oligonucleotide (competition: comp). Oligonucleotide-bound E2F1 protein was detected by western blotting using anti-flag antibody (M2). The binding capacity of each oligonucleotide is given as percentage at bottom. All panels are representative of two independent experiments.
    Gfp Topo Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Shanghai Genechem gfp express vector pgcl gfp
    EBNA3C blocks p73 and Apaf-1 expressions by inhibiting the DNA-binding ability of E2F1 to its targeted promoters in LCLs. A–B) Approximately10 million human peripheral blood mononuclear cells (PBMC) were infected by either A) wild-type (WT) BAC <t>GFP-EBV</t> or EBNA3C knockout BAC GFP-EBV (ΔE3C) for 4 h. At indicates times post-infection cells were harvested, total RNA was isolated and subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1 and EBNA3C transcript levels. Each sample was tested in triplicate and data obtained from two independent experiments with two different donors and expressed as the difference of the quantity of specific transcripts to the quantity of GAPDH transcript as control. The fold change in expression of each mRNA relative to GAPDH transcript was calculated based on the threshold cycle (Ct) as 2 − Δ(ΔCt) , where ΔCt = Ct target −Ct GAPDH and Δ(ΔCt) = ΔCt test sample −ΔCt control sample . C) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins were subjected to western blot (WB) analysis using indicated antibodies. D) Approximately 20 million of EBNA3C knockdown LCLs were <t>transfected</t> with 50 ìg of plasmids expressing either vector control or myc-tagged EBNA3C via electroporation. Transfected LCLs were cultured in RPMI medium with 10% FBS for 48 h and subjected for western blot analysis using indicated antibodies. E) Total RNA was isolated from indicated LCLs, subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1, p73 and Apaf-1 transcript levels as similar to A–B). F) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins (50 µg) were subjected to western blot analysis using indicated antibodies. G) A ChIP assay was performed using either control or EBNA3C knockdown LCLs. Material immunoprecipitated with anti-E2F1 or control antibody (rabbit IgG) was amplified by using primers specific for p73 (top) or Apaf1 (bottom) promoters. The end products of qPCR bands ran into a 2.5% agarose gel. Bar diagrams represent the change in Ct value (ΔCt) over IgG. H–I) Saos-2 (p53 −/− pRb −/− ) cells were transfected with either 5 µg of the wild-type H) p73-luciferase or I) Apaf-1-luciferase reporter plasmids with flag-E2F1 and myc-EBNA3C expression vectors as indicated. Luciferase activity was assessed at 36 h of post-transfection. J) Schematic representation of streptavidin pulldown assay as described in ‘ Materials and Methods ’ section. K) 100 µg of cell extracts from Saos-2 cells transfected with flag-E2F1 with or without myc-EBNA3C expression vector were incubated with 200 ng of the indicated biotinylated oligonucleotides (WT or Mut) immobilized with streptavidin accordingly to the manufacturer protocol, in the absence or presence of a 200 molar excess of the corresponding non biotinylated oligonucleotide (competition: comp). Oligonucleotide-bound E2F1 protein was detected by western blotting using anti-flag antibody (M2). The binding capacity of each oligonucleotide is given as percentage at bottom. All panels are representative of two independent experiments.
    Gfp Express Vector Pgcl Gfp, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 88/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp express vector pgcl gfp/product/Shanghai Genechem
    Average 88 stars, based on 54 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    Lentiviral (LV)-wild-type (WT)-HSCT corrects behavior. The open-field behavior test was performed for one hour at the same point of the circadian rhythm at 6 months (24 weeks) of age ( n = 10 female mice per group). The measures of hyperactivity were:

    Journal: Molecular Therapy

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice

    doi: 10.1038/mt.2012.82

    Figure Lengend Snippet: Lentiviral (LV)-wild-type (WT)-HSCT corrects behavior. The open-field behavior test was performed for one hour at the same point of the circadian rhythm at 6 months (24 weeks) of age ( n = 10 female mice per group). The measures of hyperactivity were:

    Article Snippet: Lentiviral vector was titred using a similar method to Kutner et al. 1 × 105 murine lymphoma (EL4) cells (ATCC Number TIB-39; Sigma-Aldrich, Gillingham, Dorset) were cultured in RPMI 1640 (Lonza)/10% FCS/2 mmol/l L -glutamine and transduced with five dilutions of concentrated lentiviral vector.

    Techniques: Mouse Assay

    Lentiviral (LV)- N -sulfoglucosamine sulfohydrolase (SGSH) improves enzyme activity in the brain of mucopolysaccharidosis IIIA (MPS IIIA) mice. ( a ) Lineage depleted bone marrow was transduced or untransduced with a lentiviral vector-expressing human SGSH

    Journal: Molecular Therapy

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice

    doi: 10.1038/mt.2012.82

    Figure Lengend Snippet: Lentiviral (LV)- N -sulfoglucosamine sulfohydrolase (SGSH) improves enzyme activity in the brain of mucopolysaccharidosis IIIA (MPS IIIA) mice. ( a ) Lineage depleted bone marrow was transduced or untransduced with a lentiviral vector-expressing human SGSH

    Article Snippet: Lentiviral vector was titred using a similar method to Kutner et al. 1 × 105 murine lymphoma (EL4) cells (ATCC Number TIB-39; Sigma-Aldrich, Gillingham, Dorset) were cultured in RPMI 1640 (Lonza)/10% FCS/2 mmol/l L -glutamine and transduced with five dilutions of concentrated lentiviral vector.

    Techniques: Activity Assay, Mouse Assay, Plasmid Preparation, Expressing

    Lentiviral (LV)- N -sulfoglucosamine sulfohydrolase (SGSH) reduces primary storage in mucopolysaccharidosis IIIA (MPS IIIA) mice. At 8 months of age the level of sulfated glycosaminoglycans was determined using the Blyscan assay in the ( a ) liver and ( b

    Journal: Molecular Therapy

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice

    doi: 10.1038/mt.2012.82

    Figure Lengend Snippet: Lentiviral (LV)- N -sulfoglucosamine sulfohydrolase (SGSH) reduces primary storage in mucopolysaccharidosis IIIA (MPS IIIA) mice. At 8 months of age the level of sulfated glycosaminoglycans was determined using the Blyscan assay in the ( a ) liver and ( b

    Article Snippet: Lentiviral vector was titred using a similar method to Kutner et al. 1 × 105 murine lymphoma (EL4) cells (ATCC Number TIB-39; Sigma-Aldrich, Gillingham, Dorset) were cultured in RPMI 1640 (Lonza)/10% FCS/2 mmol/l L -glutamine and transduced with five dilutions of concentrated lentiviral vector.

    Techniques: Mouse Assay

    Lentiviral N -sulfoglucosamine sulfohydrolase (SGSH) transduced microglia and hematopoietic stem cells (HSCs) have improved SGSH activity. ( a was converted to pHRsin.SFFV.hSGSH.att.wpre and pHRsin.SFFV.eGFP.att.wpre.

    Journal: Molecular Therapy

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice

    doi: 10.1038/mt.2012.82

    Figure Lengend Snippet: Lentiviral N -sulfoglucosamine sulfohydrolase (SGSH) transduced microglia and hematopoietic stem cells (HSCs) have improved SGSH activity. ( a was converted to pHRsin.SFFV.hSGSH.att.wpre and pHRsin.SFFV.eGFP.att.wpre.

    Article Snippet: Lentiviral vector was titred using a similar method to Kutner et al. 1 × 105 murine lymphoma (EL4) cells (ATCC Number TIB-39; Sigma-Aldrich, Gillingham, Dorset) were cultured in RPMI 1640 (Lonza)/10% FCS/2 mmol/l L -glutamine and transduced with five dilutions of concentrated lentiviral vector.

    Techniques: Activity Assay

    Lentiviral (LV)- N -sulfoglucosamine sulfohydrolase (SGSH) increases lifespan. A cohort of 6–10 mice from each group were kept to 92 weeks of age. Mice were sacrificed when they reached their humane end point, frequently caused due to urine retention.

    Journal: Molecular Therapy

    Article Title: Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice

    doi: 10.1038/mt.2012.82

    Figure Lengend Snippet: Lentiviral (LV)- N -sulfoglucosamine sulfohydrolase (SGSH) increases lifespan. A cohort of 6–10 mice from each group were kept to 92 weeks of age. Mice were sacrificed when they reached their humane end point, frequently caused due to urine retention.

    Article Snippet: Lentiviral vector was titred using a similar method to Kutner et al. 1 × 105 murine lymphoma (EL4) cells (ATCC Number TIB-39; Sigma-Aldrich, Gillingham, Dorset) were cultured in RPMI 1640 (Lonza)/10% FCS/2 mmol/l L -glutamine and transduced with five dilutions of concentrated lentiviral vector.

    Techniques: Mouse Assay

    shRNA-dependent knockdown of CaM. A , Diagram of lentiviral vectors harboring the human H1 and U6 RNA-polymerase III promoters, which drive expression of shRNAs for KD of CaM1 and CaM2 (H1) and CaM3 (U6). The vector also contains a RNA-polymerase II ubiquitin promoter (Ub) that drives expression of EGFP (to visualize infected neurons, via an IRES sequence) without or with rescue cDNAs (e.g., CaM1 without or with Ca 2+ as supplemental material). B , Analysis of CaM KD efficiency using qRT-PCR of mRNAs encoding CaM1, CaM2, and CaM3. Cultured cortical neurons were infected with control or CaM KD vectors on DIV5 and analyzed on DIV14 (means ± SEMs; numbers in bars indicate number of experiments; statistical significance was calculated by Student's t test, *** p

    Journal: The Journal of Neuroscience

    Article Title: Calmodulin Controls Synaptic Strength via Presynaptic Activation of Calmodulin Kinase II

    doi: 10.1523/JNEUROSCI.3129-09.2010

    Figure Lengend Snippet: shRNA-dependent knockdown of CaM. A , Diagram of lentiviral vectors harboring the human H1 and U6 RNA-polymerase III promoters, which drive expression of shRNAs for KD of CaM1 and CaM2 (H1) and CaM3 (U6). The vector also contains a RNA-polymerase II ubiquitin promoter (Ub) that drives expression of EGFP (to visualize infected neurons, via an IRES sequence) without or with rescue cDNAs (e.g., CaM1 without or with Ca 2+ as supplemental material). B , Analysis of CaM KD efficiency using qRT-PCR of mRNAs encoding CaM1, CaM2, and CaM3. Cultured cortical neurons were infected with control or CaM KD vectors on DIV5 and analyzed on DIV14 (means ± SEMs; numbers in bars indicate number of experiments; statistical significance was calculated by Student's t test, *** p

    Article Snippet: The lentiviral expression vector (control vector L309 or the shRNA-expressing vectors) and three helper plasmids (pRSV–REV, pMDLg/pRRE, and vesicular stomatitis virus G protein-expressing plasmid) were cotransfected with the lentiviral vectors into human embryonic kidney 293T (HEK293T) cells (American Type Culture Collection) at 4, 2, 2, and 2 μg of DNA per 25 cm2 culture area, respectively, using FUGENE 6 transfection reagent (Roche) following the instructions of the manufacturer.

    Techniques: shRNA, Chick Chorioallantoic Membrane Assay, Expressing, Plasmid Preparation, Infection, Sequencing, Quantitative RT-PCR, Cell Culture

    No postsynaptic contribution observed after CaM KD in reduction of synaptic strength. A , Image of sparsely transfected cultured cortical neurons expressing CaM shRNAs and mCherry. B , Representative traces of evoked IPSCs from cultured cortical neurons testing the effects of a postsynaptic CaM KD. Left traces depict experiments in which neurons were transfected with CaM shRNAs to produce a selective postsynaptic CaM KD in a few neurons in a culture dish, using mCherry as a marker for the transfected neurons. Right traces depict experiments in which all neurons were subjected to lentiviral CaM KD without or with lentivirally mediated WT CaM rescue, using mCherry as a marker for the infected neurons. Subsequently, the lentivirally mediated CaM KD neurons were transfected with shRNA-resistant WT CaM, such that only a few neurons were transfected that were identified by coexpressed EGFP. For all experiments, lentiviral infections were performed at DIV5, transfections at DIV9, and electrophysiological analyses at DIV14. Note that the two experimental designs are complementary, because the design depicted in the left traces measures the effect of a selective postsynaptic CaM KD, whereas the design depicted in the right traces measures the effect of a postsynaptic CaM rescue in CaM KD neurons. C , Summary graphs of evoked IPSC amplitudes in neurons that were manipulated as described in B . Data show means ± SEMs from three independent experiments; numbers in bars indicate number of cells analyzed. Statistical significance was calculated by Student's t test, *** p

    Journal: The Journal of Neuroscience

    Article Title: Calmodulin Controls Synaptic Strength via Presynaptic Activation of Calmodulin Kinase II

    doi: 10.1523/JNEUROSCI.3129-09.2010

    Figure Lengend Snippet: No postsynaptic contribution observed after CaM KD in reduction of synaptic strength. A , Image of sparsely transfected cultured cortical neurons expressing CaM shRNAs and mCherry. B , Representative traces of evoked IPSCs from cultured cortical neurons testing the effects of a postsynaptic CaM KD. Left traces depict experiments in which neurons were transfected with CaM shRNAs to produce a selective postsynaptic CaM KD in a few neurons in a culture dish, using mCherry as a marker for the transfected neurons. Right traces depict experiments in which all neurons were subjected to lentiviral CaM KD without or with lentivirally mediated WT CaM rescue, using mCherry as a marker for the infected neurons. Subsequently, the lentivirally mediated CaM KD neurons were transfected with shRNA-resistant WT CaM, such that only a few neurons were transfected that were identified by coexpressed EGFP. For all experiments, lentiviral infections were performed at DIV5, transfections at DIV9, and electrophysiological analyses at DIV14. Note that the two experimental designs are complementary, because the design depicted in the left traces measures the effect of a selective postsynaptic CaM KD, whereas the design depicted in the right traces measures the effect of a postsynaptic CaM rescue in CaM KD neurons. C , Summary graphs of evoked IPSC amplitudes in neurons that were manipulated as described in B . Data show means ± SEMs from three independent experiments; numbers in bars indicate number of cells analyzed. Statistical significance was calculated by Student's t test, *** p

    Article Snippet: The lentiviral expression vector (control vector L309 or the shRNA-expressing vectors) and three helper plasmids (pRSV–REV, pMDLg/pRRE, and vesicular stomatitis virus G protein-expressing plasmid) were cotransfected with the lentiviral vectors into human embryonic kidney 293T (HEK293T) cells (American Type Culture Collection) at 4, 2, 2, and 2 μg of DNA per 25 cm2 culture area, respectively, using FUGENE 6 transfection reagent (Roche) following the instructions of the manufacturer.

    Techniques: Chick Chorioallantoic Membrane Assay, Transfection, Cell Culture, Expressing, Marker, Infection, shRNA

    Effects of LGALS4 protein expression (gal-4) in T24 transfectants The cells were transfected with either the pCMV6-AC-GFP/gal-4 (T24/gal-4 cell line) or empty vector (T24/mock cell line) as a control. (A) Ectopic expression of gal-4 in T24 transfectants. Proteins from the whole extracts of T24/mock or T24/gal-4 cells were analyzed by Western analysis for the detection of gal-4 or β-actin (as a loading control). The lane beside the markers was run with the extracts of T24 cells as a contrast. T24/gal-4* was T24 transfectants containing the pIRES-EGFP/gal-4 vector, which showed gal-4 expression (36 kD) only as a contrast. (B) Cell proliferation of T24/mock and T24/gal-4 cells, as determined in cell viability assays using trypan blue exclusion method. Bars represent the mean± SEM of three independent experiments performed in duplicate. ** P

    Journal: Oncotarget

    Article Title: Promoter hypermethylation of LGALS4 correlates with poor prognosis in patients with urothelial carcinoma

    doi: 10.18632/oncotarget.15865

    Figure Lengend Snippet: Effects of LGALS4 protein expression (gal-4) in T24 transfectants The cells were transfected with either the pCMV6-AC-GFP/gal-4 (T24/gal-4 cell line) or empty vector (T24/mock cell line) as a control. (A) Ectopic expression of gal-4 in T24 transfectants. Proteins from the whole extracts of T24/mock or T24/gal-4 cells were analyzed by Western analysis for the detection of gal-4 or β-actin (as a loading control). The lane beside the markers was run with the extracts of T24 cells as a contrast. T24/gal-4* was T24 transfectants containing the pIRES-EGFP/gal-4 vector, which showed gal-4 expression (36 kD) only as a contrast. (B) Cell proliferation of T24/mock and T24/gal-4 cells, as determined in cell viability assays using trypan blue exclusion method. Bars represent the mean± SEM of three independent experiments performed in duplicate. ** P

    Article Snippet: The PCR product was digested and subcloned into the pCMV6-AC-GFP vector (Origene, Rockville, MD, USA) under the control of a CMV promoter.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot

    Aldolase A (ALDOA) knockdown decreased the proliferation capacity of non-small cell lung cancer (NSCLC). a Comparison of the endogenous ALDOA expression of several NSCLC cell lines. All cell lines except H157 displayed abundant expression of ALDOA. Data are shown as mean ± standard deviation (SD). b H520 cells were stably transfected with pGPU6/GFP/Neo-ALDOA-short hairpin RNA (shAL-1, shAL-2) or pGPU6/GFP/Neo-shNC (shNC, negative control). ALDOA was lower in shAL H520 cells than shNC cells. Data are shown as mean ± SD. * P

    Journal: Cancer Communications

    Article Title: Aldolase A promotes proliferation and G1/S transition via the EGFR/MAPK pathway in non-small cell lung cancer

    doi: 10.1186/s40880-018-0290-3

    Figure Lengend Snippet: Aldolase A (ALDOA) knockdown decreased the proliferation capacity of non-small cell lung cancer (NSCLC). a Comparison of the endogenous ALDOA expression of several NSCLC cell lines. All cell lines except H157 displayed abundant expression of ALDOA. Data are shown as mean ± standard deviation (SD). b H520 cells were stably transfected with pGPU6/GFP/Neo-ALDOA-short hairpin RNA (shAL-1, shAL-2) or pGPU6/GFP/Neo-shNC (shNC, negative control). ALDOA was lower in shAL H520 cells than shNC cells. Data are shown as mean ± SD. * P

    Article Snippet: A pGPU6/GFP/Neo vector carrying short hairpin RNA of ALDOA (shALDOA or shAL) or negative control sequence (shNC) (GenePharma, Suzhou, China) was transfected to H520 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, US).

    Techniques: Expressing, Standard Deviation, Stable Transfection, Transfection, shRNA, Negative Control

    Overexpression of miR-675-5p inhibits NSCLC in vivo. (A) Tumor volumes of subcutaneous implantation models of NSCLC are shown. (B) Tumor growth curves of subcutaneous implantation models of NSCLC. (C) Tumor volumes in the orthotopic implantation models at week 4 are shown. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector *P

    Journal: Molecular Cancer

    Article Title: Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

    doi: 10.1186/s12943-015-0342-0

    Figure Lengend Snippet: Overexpression of miR-675-5p inhibits NSCLC in vivo. (A) Tumor volumes of subcutaneous implantation models of NSCLC are shown. (B) Tumor growth curves of subcutaneous implantation models of NSCLC. (C) Tumor volumes in the orthotopic implantation models at week 4 are shown. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector *P

    Article Snippet: The sequence was amplified and cloned into the pGCsil-GFP Vector to generate pGCsil-GFP-miR-675-5p-inhibition.

    Techniques: Over Expression, In Vivo, Negative Control, Plasmid Preparation

    Overexpression of miR-675-5p inhibited proliferation and colony formation of NSCLC cells. (A) the level of miR-675-5p in A549 and HTB-182 cells was significantly up-regulated after transfection with miR-675-precursor. (B) miR-675-5p reduced cell proliferation in NSCLC cells. Cell proliferation was determined using MTT assays. (C) miR-675-5p induced cell cycle arrest at the G1/S phase. (D) miR-675-5p suppressed colony formation compared with controls. The number of colonies were calculated and depicted by the ban graph. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Journal: Molecular Cancer

    Article Title: Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

    doi: 10.1186/s12943-015-0342-0

    Figure Lengend Snippet: Overexpression of miR-675-5p inhibited proliferation and colony formation of NSCLC cells. (A) the level of miR-675-5p in A549 and HTB-182 cells was significantly up-regulated after transfection with miR-675-precursor. (B) miR-675-5p reduced cell proliferation in NSCLC cells. Cell proliferation was determined using MTT assays. (C) miR-675-5p induced cell cycle arrest at the G1/S phase. (D) miR-675-5p suppressed colony formation compared with controls. The number of colonies were calculated and depicted by the ban graph. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Article Snippet: The sequence was amplified and cloned into the pGCsil-GFP Vector to generate pGCsil-GFP-miR-675-5p-inhibition.

    Techniques: Over Expression, Transfection, MTT Assay, Negative Control, Plasmid Preparation

    The effect of miR-675-5p on in vitro migration and invasiveness of NSCLC cells. (A) The number of migrating or invading cells in the miR-675-precursor group was significantly decreased compared with the control. (B) The wound healing rate in cells transfected with miR-675-precursor was significantly decreased. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Journal: Molecular Cancer

    Article Title: Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

    doi: 10.1186/s12943-015-0342-0

    Figure Lengend Snippet: The effect of miR-675-5p on in vitro migration and invasiveness of NSCLC cells. (A) The number of migrating or invading cells in the miR-675-precursor group was significantly decreased compared with the control. (B) The wound healing rate in cells transfected with miR-675-precursor was significantly decreased. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Article Snippet: The sequence was amplified and cloned into the pGCsil-GFP Vector to generate pGCsil-GFP-miR-675-5p-inhibition.

    Techniques: In Vitro, Migration, Transfection, Negative Control, Plasmid Preparation

    GPR55 is a direct downstream target of miR-675-5p. (A) Schematic of the construction of wild-type or mutant pGL3-GPR55 3′-UTR vectors is illustrated. (B) Relative luciferase activity was analyzed in A549 cells. Firefly luciferase vector was used as an internal control. (C and D) Related expression of GPR55 protein in A549 and HTB-182 cells treated with miR-675 precursor was determined by western blot analysis. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Journal: Molecular Cancer

    Article Title: Down-regulation of miR-675-5p contributes to tumor progression and development by targeting pro-tumorigenic GPR55 in non-small cell lung cancer

    doi: 10.1186/s12943-015-0342-0

    Figure Lengend Snippet: GPR55 is a direct downstream target of miR-675-5p. (A) Schematic of the construction of wild-type or mutant pGL3-GPR55 3′-UTR vectors is illustrated. (B) Relative luciferase activity was analyzed in A549 cells. Firefly luciferase vector was used as an internal control. (C and D) Related expression of GPR55 protein in A549 and HTB-182 cells treated with miR-675 precursor was determined by western blot analysis. Data were represented as the mean ± SEM of three independent experiments. Negative control: pGCsil-GFP Vector. *P

    Article Snippet: The sequence was amplified and cloned into the pGCsil-GFP Vector to generate pGCsil-GFP-miR-675-5p-inhibition.

    Techniques: Mutagenesis, Luciferase, Activity Assay, Plasmid Preparation, Expressing, Western Blot, Negative Control

    Studies of the minimum Speedy A fragment that is required for its localization to telomeres. The different regions of Speedy A cDNA were cloned into the pCAG-GFP vector and expressed by electroporation in wild-type testes. ( A ) Western blot showing that

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Speedy A–Cdk2 binding mediates initial telomere–nuclear envelope attachment during meiotic prophase I independent of Cdk2 activation

    doi: 10.1073/pnas.1618465114

    Figure Lengend Snippet: Studies of the minimum Speedy A fragment that is required for its localization to telomeres. The different regions of Speedy A cDNA were cloned into the pCAG-GFP vector and expressed by electroporation in wild-type testes. ( A ) Western blot showing that

    Article Snippet: Different fragments of Spdya were cloned into the pCAG-GFP vector (Addgene #11150).

    Techniques: Clone Assay, Plasmid Preparation, Electroporation, Western Blot

    Localization of Cdk2 on telomeres is mediated by its interaction with Speedy A. ( A ) p39 Cdk2 cDNAs carrying point mutations were cloned into the pCAG-GFP vector and expressed in 293T cells, a total of 30-μg cell lysate was used for Western blot

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Speedy A–Cdk2 binding mediates initial telomere–nuclear envelope attachment during meiotic prophase I independent of Cdk2 activation

    doi: 10.1073/pnas.1618465114

    Figure Lengend Snippet: Localization of Cdk2 on telomeres is mediated by its interaction with Speedy A. ( A ) p39 Cdk2 cDNAs carrying point mutations were cloned into the pCAG-GFP vector and expressed in 293T cells, a total of 30-μg cell lysate was used for Western blot

    Article Snippet: Different fragments of Spdya were cloned into the pCAG-GFP vector (Addgene #11150).

    Techniques: Clone Assay, Plasmid Preparation, Western Blot

    GMFG overexpression enhances chemoattractant-stimulated cell migration and adhesion to FN. Human monocytes or THP-1 cells were transfected with GFP vector or GFP-tagged GMFG plasmid for 48 h. A , Western blotting analysis of expression of GMFG or GMFG-GFP

    Journal: The Journal of Biological Chemistry

    Article Title: Glia Maturation Factor-γ Regulates Monocyte Migration through Modulation of β1-Integrin *

    doi: 10.1074/jbc.M115.674200

    Figure Lengend Snippet: GMFG overexpression enhances chemoattractant-stimulated cell migration and adhesion to FN. Human monocytes or THP-1 cells were transfected with GFP vector or GFP-tagged GMFG plasmid for 48 h. A , Western blotting analysis of expression of GMFG or GMFG-GFP

    Article Snippet: GFP vector and GMFG-GFP plasmid (human cDNA clone, ORF with C-terminal GFP tag) were obtained from ORIGENE.

    Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Western Blot, Expressing

    Targeting of angiogenic ECs with CD44 or EphA2 siRNA blocks in vivo LMW-HA-mediated angiogenesis. Panel A , Matrigel plugs from mice with intravenous administration of anginex-conjugated liposomes containing either pCMV6 empty vector or pCMV6-A-GFP (Origene)

    Journal: The Journal of Biological Chemistry

    Article Title: Transactivation of the Receptor-tyrosine Kinase Ephrin Receptor A2 Is Required for the Low Molecular Weight Hyaluronan-mediated Angiogenesis That Is implicated in Tumor Progression *

    doi: 10.1074/jbc.M114.554766

    Figure Lengend Snippet: Targeting of angiogenic ECs with CD44 or EphA2 siRNA blocks in vivo LMW-HA-mediated angiogenesis. Panel A , Matrigel plugs from mice with intravenous administration of anginex-conjugated liposomes containing either pCMV6 empty vector or pCMV6-A-GFP (Origene)

    Article Snippet: Sterile anginex-conjugated liposomes containing no siRNA (control), CD44, EphA2, or control siRNA (100 μl) or pCMV6 empty vector or GFP expression vector (pCMV6-A-GFP Vector, Origene) were injected into the internal jugular vein of C57B6/6J mice.

    Techniques: In Vivo, Mouse Assay, Plasmid Preparation

    Downregulation of the α2δ1 subunit impairs the presynaptic release of glutamate and abolishes α2δ1 overexpression-driven enhancement of spontaneous neuronal firing. A , B , shRNA-induced knockdown of the α2δ1 subunit results in significant decrease of its surface expression and in corresponding decrease of the live HA fluorescence in rat hippocampal neurons. C , Western blot demonstrates a significant decrease in neuronal expression of the α2δ1 subunit on shRNA-triggered knockdown. D , A timeline of infection (green triangle) and electrophysiological recordings (orange triangles) shown in E–H . E , Downregulation of the α2δ1 subunit, but not the GFP expression, leads to significant reduction of the mean frequency of mEPSCs in rat hippocampal neurons. F , Cumulative distribution of interevent intervals for mEPSCs recorded under control conditions or on α2δ1 knockdown. G , The mean mEPSC amplitude is not affected by either α2δ1 knockdown, or by lentiviral expression of the GFP. H , Cumulative distribution of mEPSC amplitudes recorded under control conditions or on α2δ1 knockdown. I , A timeline of infection (green triangle) and multichannel recordings (orange triangles) shown in J , K . J , Representative traces of spontaneous neuronal firing in rat hippocampal cultures under control conditions (black), as well as after 1 week of either α2δ1 overexpression (red) or knockdown (brown). Thirty of 60 channels from each array are shown. Scale bar, 10 s. K , The shRNA-mediated knockdown of the α2δ1 subunit during the fourth week in vitro is associated with suppression of the spontaneous neuronal firing. * p

    Journal: The Journal of Neuroscience

    Article Title: Auxiliary α2δ1 and α2δ3 Subunits of Calcium Channels Drive Excitatory and Inhibitory Neuronal Network Development

    doi: 10.1523/JNEUROSCI.1707-19.2020

    Figure Lengend Snippet: Downregulation of the α2δ1 subunit impairs the presynaptic release of glutamate and abolishes α2δ1 overexpression-driven enhancement of spontaneous neuronal firing. A , B , shRNA-induced knockdown of the α2δ1 subunit results in significant decrease of its surface expression and in corresponding decrease of the live HA fluorescence in rat hippocampal neurons. C , Western blot demonstrates a significant decrease in neuronal expression of the α2δ1 subunit on shRNA-triggered knockdown. D , A timeline of infection (green triangle) and electrophysiological recordings (orange triangles) shown in E–H . E , Downregulation of the α2δ1 subunit, but not the GFP expression, leads to significant reduction of the mean frequency of mEPSCs in rat hippocampal neurons. F , Cumulative distribution of interevent intervals for mEPSCs recorded under control conditions or on α2δ1 knockdown. G , The mean mEPSC amplitude is not affected by either α2δ1 knockdown, or by lentiviral expression of the GFP. H , Cumulative distribution of mEPSC amplitudes recorded under control conditions or on α2δ1 knockdown. I , A timeline of infection (green triangle) and multichannel recordings (orange triangles) shown in J , K . J , Representative traces of spontaneous neuronal firing in rat hippocampal cultures under control conditions (black), as well as after 1 week of either α2δ1 overexpression (red) or knockdown (brown). Thirty of 60 channels from each array are shown. Scale bar, 10 s. K , The shRNA-mediated knockdown of the α2δ1 subunit during the fourth week in vitro is associated with suppression of the spontaneous neuronal firing. * p

    Article Snippet: For knockdown of the α2δ3 subunit, four 29mer shRNA constructs against rat Cacna2d3 (Gene ID 306243) cloned in lentiviral GFP vector (pGFP-C-shLenti Vector, catalog #TR30023) were ordered from OriGene Technologies (catalog #TL713428).

    Techniques: Over Expression, shRNA, Expressing, Fluorescence, Western Blot, Infection, In Vitro

    Characterization of HA-tagged α2δ1 and α2δ3 subunits and protein expression levels before and after lentiviral-induced overexpression. A , Schemes of double HA-tagged α2δ1 (left) and α2δ3 (right) subunits. Purple represents the localization of the HA tag. B , C , Validation of the α2δ1 antibodies in Western blots of either untreated HEK293T cells (control, ctrl) or HEK293T cells expressing the α2δ1-HA ( A ) or α2δ3-HA ( B ) subunit. The HA-tagged α2δ proteins were detected using either the anti-α2δ antibodies (left) or a highly specific anti-HA antibody that served as positive control (right). Validation of the α2δ antibodies in live immunocytochemical stainings of DIV16 hippocampal cultures expressing the HA-tagged α2δ subunits and GFP to identify transfected cells. Scale bars, 5 µm. D , Representative images of neuronal cultures at DIV16 stained against the HA tag (live, green), GFAP (magenta), and DAPI (blue) to show α2δ1-HA-infected neurons, glial cells, and the total cell number, respectively. Glial cells do not express the α2δ1 subunit, thus confirming neuron-specific expression. Scale bar, 50 μm. E , F , Exemplary Western blots showing the endogenous (ctrl) and viral-boosted expression of α2δ1 ( E ) or α2δ3 ( F ) in neurons at DIV16. G , H , Lentiviral infection significantly increases total protein level of the α2δ1 ( G ) and α2δ3 ( H ) subunits. GFAP, glial fibrillary acidic protein, DAPI, 4′,6-diamidino-2-phenylindole. * p

    Journal: The Journal of Neuroscience

    Article Title: Auxiliary α2δ1 and α2δ3 Subunits of Calcium Channels Drive Excitatory and Inhibitory Neuronal Network Development

    doi: 10.1523/JNEUROSCI.1707-19.2020

    Figure Lengend Snippet: Characterization of HA-tagged α2δ1 and α2δ3 subunits and protein expression levels before and after lentiviral-induced overexpression. A , Schemes of double HA-tagged α2δ1 (left) and α2δ3 (right) subunits. Purple represents the localization of the HA tag. B , C , Validation of the α2δ1 antibodies in Western blots of either untreated HEK293T cells (control, ctrl) or HEK293T cells expressing the α2δ1-HA ( A ) or α2δ3-HA ( B ) subunit. The HA-tagged α2δ proteins were detected using either the anti-α2δ antibodies (left) or a highly specific anti-HA antibody that served as positive control (right). Validation of the α2δ antibodies in live immunocytochemical stainings of DIV16 hippocampal cultures expressing the HA-tagged α2δ subunits and GFP to identify transfected cells. Scale bars, 5 µm. D , Representative images of neuronal cultures at DIV16 stained against the HA tag (live, green), GFAP (magenta), and DAPI (blue) to show α2δ1-HA-infected neurons, glial cells, and the total cell number, respectively. Glial cells do not express the α2δ1 subunit, thus confirming neuron-specific expression. Scale bar, 50 μm. E , F , Exemplary Western blots showing the endogenous (ctrl) and viral-boosted expression of α2δ1 ( E ) or α2δ3 ( F ) in neurons at DIV16. G , H , Lentiviral infection significantly increases total protein level of the α2δ1 ( G ) and α2δ3 ( H ) subunits. GFAP, glial fibrillary acidic protein, DAPI, 4′,6-diamidino-2-phenylindole. * p

    Article Snippet: For knockdown of the α2δ3 subunit, four 29mer shRNA constructs against rat Cacna2d3 (Gene ID 306243) cloned in lentiviral GFP vector (pGFP-C-shLenti Vector, catalog #TR30023) were ordered from OriGene Technologies (catalog #TL713428).

    Techniques: Expressing, Over Expression, Western Blot, Positive Control, Transfection, Staining, Infection

    Overexpression of the α2δ3 subunit during the first week in vitro promotes axonal outgrowth and branching in young interneurons. A-C , Representative images of GAD67::GFP mouse hippocampal neurons at DIV9 in control conditions ( A ), as well as after lentiviral infection at DIV2-DIV4 with either pLenti-syn-α2δ1::HA ( B ) or pLenti-syn-α2δ3::HA ( C ). The length and the branching of axons were analyzed exclusively in GAD67-positive interneurons (arrows), which were identified among other neurons (arrowheads) by GFP immunofluorescence. Scale bars, 50 µm. D , Upregulation of the α2δ3, but not α2δ1, subunit promotes the axonal outgrowth in GAD67-positive interneurons compared with controls. E , Overexpression of α2δ3 during the first developmental week leads to twofold increase in axonal branching compared with α2δ1-overexpressing or control cultures. GAD67, glutamic acid decarboxylase isoform 67, MAP2, microtubule-associated protein 2. *** p

    Journal: The Journal of Neuroscience

    Article Title: Auxiliary α2δ1 and α2δ3 Subunits of Calcium Channels Drive Excitatory and Inhibitory Neuronal Network Development

    doi: 10.1523/JNEUROSCI.1707-19.2020

    Figure Lengend Snippet: Overexpression of the α2δ3 subunit during the first week in vitro promotes axonal outgrowth and branching in young interneurons. A-C , Representative images of GAD67::GFP mouse hippocampal neurons at DIV9 in control conditions ( A ), as well as after lentiviral infection at DIV2-DIV4 with either pLenti-syn-α2δ1::HA ( B ) or pLenti-syn-α2δ3::HA ( C ). The length and the branching of axons were analyzed exclusively in GAD67-positive interneurons (arrows), which were identified among other neurons (arrowheads) by GFP immunofluorescence. Scale bars, 50 µm. D , Upregulation of the α2δ3, but not α2δ1, subunit promotes the axonal outgrowth in GAD67-positive interneurons compared with controls. E , Overexpression of α2δ3 during the first developmental week leads to twofold increase in axonal branching compared with α2δ1-overexpressing or control cultures. GAD67, glutamic acid decarboxylase isoform 67, MAP2, microtubule-associated protein 2. *** p

    Article Snippet: For knockdown of the α2δ3 subunit, four 29mer shRNA constructs against rat Cacna2d3 (Gene ID 306243) cloned in lentiviral GFP vector (pGFP-C-shLenti Vector, catalog #TR30023) were ordered from OriGene Technologies (catalog #TL713428).

    Techniques: Over Expression, In Vitro, Infection, Immunofluorescence

    Downregulation of the α2δ3 subunit impairs spontaneous GABA release and leads to suppression of neuronal firing in developing hippocampal neurons. A , B , The shRNA-mediated knockdown of the α2δ3 subunit results in a significant decrease of the α2δ3 surface expression in HEK293T was examined via fluorescence labeling of HA-tagged α2δ3 subunits in HEK293T cells ( A , B ), compared with the effect of scrambled (scr) shRNA. Scale bar, 20 µm. C , D , Downregulation of the α2δ3 subunit in rat hippocampal neurons. Scale bar, 20 µm. E , F , Western blots of HEK293T cells expressing the HA-tagged α2δ3 subunit together with the scrambled shRNA control or the α2δ3 shRNA. G-I , Western blots of hippocampal cultures infected with the HA-tagged α2δ3 construct ( G ) or with the scrambled shRNA control, as well as the α2δ3 shRNA (blue) ( H , I ). J , A timeline of infection (green triangle) and electrophysiological recordings (orange triangles) shown in K , N . K , Downregulation of the α2δ3 subunit, but not the GFP expression, significantly decreases the mean mIPSC frequency in rat hippocampal neurons. L . Cumulative distribution of interevent intervals for mEPSCs recorded under control conditions or on α2δ1 knockdown. M , The mean mEPSC amplitude is not affected by either α2δ3 knockdown or by lentiviral expression of the GFP. N , Cumulative distribution of mEPSC amplitudes recorded under control conditions or on α2δ1 knockdown. O , A timeline of infection (green triangle) and multichannel recordings (orange triangles) shown in P , Q . P , Representative traces of spontaneous neuronal firing in rat hippocampal cultures under control conditions (black), as well as after 1 week of either α2δ3 overexpression (blue) or α2δ3 knockdown (petrol). Thirty of 60 channels from each array are shown. Scale bar, 20 s. Q , The α2δ3 overexpression in young neurons strongly enhances neuronal activity, whereas the shRNA-mediated α2δ3 knockdown leads to dramatic suppression of the mean firing rate. * p

    Journal: The Journal of Neuroscience

    Article Title: Auxiliary α2δ1 and α2δ3 Subunits of Calcium Channels Drive Excitatory and Inhibitory Neuronal Network Development

    doi: 10.1523/JNEUROSCI.1707-19.2020

    Figure Lengend Snippet: Downregulation of the α2δ3 subunit impairs spontaneous GABA release and leads to suppression of neuronal firing in developing hippocampal neurons. A , B , The shRNA-mediated knockdown of the α2δ3 subunit results in a significant decrease of the α2δ3 surface expression in HEK293T was examined via fluorescence labeling of HA-tagged α2δ3 subunits in HEK293T cells ( A , B ), compared with the effect of scrambled (scr) shRNA. Scale bar, 20 µm. C , D , Downregulation of the α2δ3 subunit in rat hippocampal neurons. Scale bar, 20 µm. E , F , Western blots of HEK293T cells expressing the HA-tagged α2δ3 subunit together with the scrambled shRNA control or the α2δ3 shRNA. G-I , Western blots of hippocampal cultures infected with the HA-tagged α2δ3 construct ( G ) or with the scrambled shRNA control, as well as the α2δ3 shRNA (blue) ( H , I ). J , A timeline of infection (green triangle) and electrophysiological recordings (orange triangles) shown in K , N . K , Downregulation of the α2δ3 subunit, but not the GFP expression, significantly decreases the mean mIPSC frequency in rat hippocampal neurons. L . Cumulative distribution of interevent intervals for mEPSCs recorded under control conditions or on α2δ1 knockdown. M , The mean mEPSC amplitude is not affected by either α2δ3 knockdown or by lentiviral expression of the GFP. N , Cumulative distribution of mEPSC amplitudes recorded under control conditions or on α2δ1 knockdown. O , A timeline of infection (green triangle) and multichannel recordings (orange triangles) shown in P , Q . P , Representative traces of spontaneous neuronal firing in rat hippocampal cultures under control conditions (black), as well as after 1 week of either α2δ3 overexpression (blue) or α2δ3 knockdown (petrol). Thirty of 60 channels from each array are shown. Scale bar, 20 s. Q , The α2δ3 overexpression in young neurons strongly enhances neuronal activity, whereas the shRNA-mediated α2δ3 knockdown leads to dramatic suppression of the mean firing rate. * p

    Article Snippet: For knockdown of the α2δ3 subunit, four 29mer shRNA constructs against rat Cacna2d3 (Gene ID 306243) cloned in lentiviral GFP vector (pGFP-C-shLenti Vector, catalog #TR30023) were ordered from OriGene Technologies (catalog #TL713428).

    Techniques: shRNA, Expressing, Fluorescence, Labeling, Western Blot, Infection, Construct, Over Expression, Activity Assay

    TRPV1-WT but not the LWI mutants co-localize with endogenous lipid raft markers. TRPV1-WT-GFP (green) and different LWI mutants in GFP (green) were expressed in F11 cells. Cells were fixed 36 hours post transfection and stained for lipid raft with Cholera Toxin B-594 (red). All images were acquired by confocal microscope. Same experiment was performed with cells that were treated with 5mM MβCD for cholesterol depletion 30 min before cell fixation. TRPV1-WT-GFP shows distinct co-localization with Cholera Toxin B in the membranous region while LWI mutants are distinctly excluded from Cholera Toxin B-enriched membranous regions.

    Journal: bioRxiv

    Article Title: Ratio of hydrophobic-hydrophilic and positive-negative residues at lipid-water-interface influences surface expression and channel-gating of TRPV1

    doi: 10.1101/2020.09.04.272484

    Figure Lengend Snippet: TRPV1-WT but not the LWI mutants co-localize with endogenous lipid raft markers. TRPV1-WT-GFP (green) and different LWI mutants in GFP (green) were expressed in F11 cells. Cells were fixed 36 hours post transfection and stained for lipid raft with Cholera Toxin B-594 (red). All images were acquired by confocal microscope. Same experiment was performed with cells that were treated with 5mM MβCD for cholesterol depletion 30 min before cell fixation. TRPV1-WT-GFP shows distinct co-localization with Cholera Toxin B in the membranous region while LWI mutants are distinctly excluded from Cholera Toxin B-enriched membranous regions.

    Article Snippet: After mutagenesis, full-length rTRPV1-WT and all LWI mutants were cloned into pSGFP2-C1 or GFP vector (Addgene) using 5′-CCAGGAATTCTATGGAACAACGGGCTAGC-3′ and 5′-CCAGGTCGACTTATTTCTCCCCTGGGACC-3′ primer sets having EcoR1 and SalI site respectively.

    Techniques: Transfection, Staining, Microscopy

    Arg residues at the Lipid-Water-Interface (LWI) of TRPV1 are required for its proper surface expression and membrane localization. The localization pattern of rTRPV1-WT-GFP or different LWI mutants in GFP has been shown. These GFP-tagged proteins (green) were transiently expressed in F-11 cells for 36 hours, fixed and imaged using confocal imaging. WT-rTRPV1 shows distinct membrane localization, whereas the LWI mutants fail to localize at the membrane. Often the LWI mutants are retained in the ER and/or cause fragmentation of ER. The intensity of GFP-tagged proteins are shown in the rainbow scale. Nucleus is stained with DAPI (blue). Enlarged view of surface areas (indicated by dotted square) for GFP fluorescence at the membrane merged with DIC image are shown in the right side.

    Journal: bioRxiv

    Article Title: Ratio of hydrophobic-hydrophilic and positive-negative residues at lipid-water-interface influences surface expression and channel-gating of TRPV1

    doi: 10.1101/2020.09.04.272484

    Figure Lengend Snippet: Arg residues at the Lipid-Water-Interface (LWI) of TRPV1 are required for its proper surface expression and membrane localization. The localization pattern of rTRPV1-WT-GFP or different LWI mutants in GFP has been shown. These GFP-tagged proteins (green) were transiently expressed in F-11 cells for 36 hours, fixed and imaged using confocal imaging. WT-rTRPV1 shows distinct membrane localization, whereas the LWI mutants fail to localize at the membrane. Often the LWI mutants are retained in the ER and/or cause fragmentation of ER. The intensity of GFP-tagged proteins are shown in the rainbow scale. Nucleus is stained with DAPI (blue). Enlarged view of surface areas (indicated by dotted square) for GFP fluorescence at the membrane merged with DIC image are shown in the right side.

    Article Snippet: After mutagenesis, full-length rTRPV1-WT and all LWI mutants were cloned into pSGFP2-C1 or GFP vector (Addgene) using 5′-CCAGGAATTCTATGGAACAACGGGCTAGC-3′ and 5′-CCAGGTCGACTTATTTCTCCCCTGGGACC-3′ primer sets having EcoR1 and SalI site respectively.

    Techniques: Expressing, Imaging, Staining, Fluorescence

    TRPV1-WT but not the Lipid Water Interface (LWI) mutants co-localize with overexpressed lipid raft markers. GFP-tagged (green) TRPV1 wild type (WT) and different LWI mutants were co-expressed with lipid raft marker Flotillin1-RFP (red) in F11 cells. Cells were fixed 36 hours post transfection and images were acquired by confocal microscope. TRPV1-WT shows distinct co-localization with Flotillin1-RFP in the membranous region. Notably, the LWI mutants are distinctly excluded from Flotillin1-RFP enriched membranous regions even after over expressing both.

    Journal: bioRxiv

    Article Title: Ratio of hydrophobic-hydrophilic and positive-negative residues at lipid-water-interface influences surface expression and channel-gating of TRPV1

    doi: 10.1101/2020.09.04.272484

    Figure Lengend Snippet: TRPV1-WT but not the Lipid Water Interface (LWI) mutants co-localize with overexpressed lipid raft markers. GFP-tagged (green) TRPV1 wild type (WT) and different LWI mutants were co-expressed with lipid raft marker Flotillin1-RFP (red) in F11 cells. Cells were fixed 36 hours post transfection and images were acquired by confocal microscope. TRPV1-WT shows distinct co-localization with Flotillin1-RFP in the membranous region. Notably, the LWI mutants are distinctly excluded from Flotillin1-RFP enriched membranous regions even after over expressing both.

    Article Snippet: After mutagenesis, full-length rTRPV1-WT and all LWI mutants were cloned into pSGFP2-C1 or GFP vector (Addgene) using 5′-CCAGGAATTCTATGGAACAACGGGCTAGC-3′ and 5′-CCAGGTCGACTTATTTCTCCCCTGGGACC-3′ primer sets having EcoR1 and SalI site respectively.

    Techniques: Marker, Transfection, Microscopy, Expressing

    TRPV1-WT but not the Lipid Water Interface (LWI) mutants co-localize with overexpressed lipid raft markers. GFP-tagged (green) TRPV1 wild type (WT) and different LWI mutants were co-expressed with lipid raft marker Caveolin1-RFP (red) in F11 cells. Cells were fixed 36 hours post transfection and images were acquired by confocal microscope. TRPV1-WT shows distinct co-localization with Caveolin1-RFP in the membranous region. Notably, the LWI mutants are distinctly excluded from Caveolin1-RFP enriched membranous regions even after over expressing both.

    Journal: bioRxiv

    Article Title: Ratio of hydrophobic-hydrophilic and positive-negative residues at lipid-water-interface influences surface expression and channel-gating of TRPV1

    doi: 10.1101/2020.09.04.272484

    Figure Lengend Snippet: TRPV1-WT but not the Lipid Water Interface (LWI) mutants co-localize with overexpressed lipid raft markers. GFP-tagged (green) TRPV1 wild type (WT) and different LWI mutants were co-expressed with lipid raft marker Caveolin1-RFP (red) in F11 cells. Cells were fixed 36 hours post transfection and images were acquired by confocal microscope. TRPV1-WT shows distinct co-localization with Caveolin1-RFP in the membranous region. Notably, the LWI mutants are distinctly excluded from Caveolin1-RFP enriched membranous regions even after over expressing both.

    Article Snippet: After mutagenesis, full-length rTRPV1-WT and all LWI mutants were cloned into pSGFP2-C1 or GFP vector (Addgene) using 5′-CCAGGAATTCTATGGAACAACGGGCTAGC-3′ and 5′-CCAGGTCGACTTATTTCTCCCCTGGGACC-3′ primer sets having EcoR1 and SalI site respectively.

    Techniques: Marker, Transfection, Microscopy, Expressing

    Immunophenotypic characterization of human MSC and MSC-mediated increase in invasiveness of pancreatic cancer cells. A. FACS analysis of human MSC using anti-human CD34, CD45, CD73, and CD105. Mouse IgG as classmatch control for mouse monoclonal CD34, CD45, rabbit IgG for rabbit monoclonal CD73 and CD105. Indirect immunofluorescence using Alexa488-conjugated secondary antibodies. B and C. BxPC3 invasiveness was assessed under the co-culture condition with hMSCs by employing Boyden chamber method. Filter membrane set in the top chamber was sealed with Matrigel to close the micro pore. The mixed cells (BxPC3-GFP cells and hMSCs, ratio 1:1) were placed in the top chamber filled with serum free medium and serum was added or not added to the bottom well to be final concentrations 0.5% and 10%. After incubation for 12 hours, passed BxPC3-GFP cells through the filter membrane were observed by the fluorescence microscope (A) and their fluorescence intensities were measured and quantified (B). Data from A are means ± SD, *P

    Journal: Journal of Cancer

    Article Title: Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction

    doi: 10.7150/jca.24415

    Figure Lengend Snippet: Immunophenotypic characterization of human MSC and MSC-mediated increase in invasiveness of pancreatic cancer cells. A. FACS analysis of human MSC using anti-human CD34, CD45, CD73, and CD105. Mouse IgG as classmatch control for mouse monoclonal CD34, CD45, rabbit IgG for rabbit monoclonal CD73 and CD105. Indirect immunofluorescence using Alexa488-conjugated secondary antibodies. B and C. BxPC3 invasiveness was assessed under the co-culture condition with hMSCs by employing Boyden chamber method. Filter membrane set in the top chamber was sealed with Matrigel to close the micro pore. The mixed cells (BxPC3-GFP cells and hMSCs, ratio 1:1) were placed in the top chamber filled with serum free medium and serum was added or not added to the bottom well to be final concentrations 0.5% and 10%. After incubation for 12 hours, passed BxPC3-GFP cells through the filter membrane were observed by the fluorescence microscope (A) and their fluorescence intensities were measured and quantified (B). Data from A are means ± SD, *P

    Article Snippet: BxPC3 cells (ATCC, Manassas, VA) and their GFP expressing subline in a stable manner, named BxPC3-GFP cells, which we established by using GFP expression vector (pEF/myc/cyto/GFP; Thermo Fisher Scientific, Waltham, MA) were cultivated in RPMI medium (Wako Laboratory Chemicals, Japan) supplemented with 10% FBS.

    Techniques: FACS, Immunofluorescence, Co-Culture Assay, Incubation, Fluorescence, Microscopy

    Gene expression profiling of the selected secretory candidates in co-culture of pancreatic cancer cells with MSCs. A and B. Total RNAs prepared from indicated cell cultures in the panel A (BxPC3 cells alone, hMSCs alone, BxPC3 cells and hMSCs (ratio 1:1 and 1:2)) and the panel B (BxPC3 cells alone, NHDFs alone, BxPC3 cells and NHDFs (ratio 1:1 and 1:2)) were analyzed for expression of the selected genes of our interest by quantitative real-time PCR. GAPDH was used as control for the analysis. Relative expression of each culture was calculated and shown by ratio (aimed genes/GADH) against that of BxPC3 cells as a standard. C. MMP-3 and MMP-9 was evaluated by the immunofluorescence staining in the co-culture system with BxPC3-GFP cells and hMSCs. BxPC3-GFP cells were detected by their own GFP green fluorescence color, while MMP-3 and MMP-9 were detected by red fluorescence colors after the immunostaining.

    Journal: Journal of Cancer

    Article Title: Stromal mesenchymal stem cells facilitate pancreatic cancer progression by regulating specific secretory molecules through mutual cellular interaction

    doi: 10.7150/jca.24415

    Figure Lengend Snippet: Gene expression profiling of the selected secretory candidates in co-culture of pancreatic cancer cells with MSCs. A and B. Total RNAs prepared from indicated cell cultures in the panel A (BxPC3 cells alone, hMSCs alone, BxPC3 cells and hMSCs (ratio 1:1 and 1:2)) and the panel B (BxPC3 cells alone, NHDFs alone, BxPC3 cells and NHDFs (ratio 1:1 and 1:2)) were analyzed for expression of the selected genes of our interest by quantitative real-time PCR. GAPDH was used as control for the analysis. Relative expression of each culture was calculated and shown by ratio (aimed genes/GADH) against that of BxPC3 cells as a standard. C. MMP-3 and MMP-9 was evaluated by the immunofluorescence staining in the co-culture system with BxPC3-GFP cells and hMSCs. BxPC3-GFP cells were detected by their own GFP green fluorescence color, while MMP-3 and MMP-9 were detected by red fluorescence colors after the immunostaining.

    Article Snippet: BxPC3 cells (ATCC, Manassas, VA) and their GFP expressing subline in a stable manner, named BxPC3-GFP cells, which we established by using GFP expression vector (pEF/myc/cyto/GFP; Thermo Fisher Scientific, Waltham, MA) were cultivated in RPMI medium (Wako Laboratory Chemicals, Japan) supplemented with 10% FBS.

    Techniques: Expressing, Co-Culture Assay, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Fluorescence, Immunostaining

    Homing of MSCs in pancreas. (A) Primary culture of MSCs showing many spindle-shaped stem cells (white arrows) ×200. (B) Fluorescent microscopic image of a section in pancreas of rat in group III demonstrating the green fluorescence of MSCs labeled with GFP two week after implantation (white arrows) ×200. (C) Flow cytometric chart analysis for surface antigens of MSCs. They were positive for CD29 and CD90.

    Journal: International Journal of Stem Cells

    Article Title: Combination of Obestatin and Bone Marrow Mesenchymal Stem Cells Prevents Aggravation of Endocrine Pancreatic Damage in Type II Diabetic Rats

    doi: 10.15283/ijsc17035

    Figure Lengend Snippet: Homing of MSCs in pancreas. (A) Primary culture of MSCs showing many spindle-shaped stem cells (white arrows) ×200. (B) Fluorescent microscopic image of a section in pancreas of rat in group III demonstrating the green fluorescence of MSCs labeled with GFP two week after implantation (white arrows) ×200. (C) Flow cytometric chart analysis for surface antigens of MSCs. They were positive for CD29 and CD90.

    Article Snippet: Green fluorescent protein (GFP) labeling of MSCs MSCs were labeled in vitro with GFP (pAcGFP1-N1 Vector, Clontech Laboratories, Inc. (USA) catalog #632469) for in vivo tracing and observed in unstained pancreatic tissues cryosections using Fluorescence Microscope (Leica Microsystems CMS GmbH, Ernst-Leitz-Straße, Wetzlar, D-35578, Germany) ( ).

    Techniques: Fluorescence, Labeling, Flow Cytometry

    Photomicrographs sections (immunohistochemistry) from rat tail pancreas showing. (A) GFP expression in transplanted MSCs. (B) CD105 expression in transplanted MSCs.

    Journal: International Journal of Stem Cells

    Article Title: Combination of Obestatin and Bone Marrow Mesenchymal Stem Cells Prevents Aggravation of Endocrine Pancreatic Damage in Type II Diabetic Rats

    doi: 10.15283/ijsc17035

    Figure Lengend Snippet: Photomicrographs sections (immunohistochemistry) from rat tail pancreas showing. (A) GFP expression in transplanted MSCs. (B) CD105 expression in transplanted MSCs.

    Article Snippet: Green fluorescent protein (GFP) labeling of MSCs MSCs were labeled in vitro with GFP (pAcGFP1-N1 Vector, Clontech Laboratories, Inc. (USA) catalog #632469) for in vivo tracing and observed in unstained pancreatic tissues cryosections using Fluorescence Microscope (Leica Microsystems CMS GmbH, Ernst-Leitz-Straße, Wetzlar, D-35578, Germany) ( ).

    Techniques: Immunohistochemistry, Expressing

    Flk-1 shRNA in U87 cells inhibits tumor growth and VM. A, Flk-1 gene knockdown results in suppression of tumor growth. U87 cells expressing Flk-1 shRNA-GFP or GFP vector were injected subcutaneously into SCID/Beige mice, and tumor size was measured weekly

    Journal: The Journal of Biological Chemistry

    Article Title: Glioblastoma-derived Tumor Cells Induce Vasculogenic Mimicry through Flk-1 Protein Activation *

    doi: 10.1074/jbc.M111.334540

    Figure Lengend Snippet: Flk-1 shRNA in U87 cells inhibits tumor growth and VM. A, Flk-1 gene knockdown results in suppression of tumor growth. U87 cells expressing Flk-1 shRNA-GFP or GFP vector were injected subcutaneously into SCID/Beige mice, and tumor size was measured weekly

    Article Snippet: A PGPU6-GFP-neo shRNA expression vector containing DNA oligonucleotides (21 bp) (GenePharma, Shanghai, China) specifically targeting the C terminus (5′-GCTTGGCCCGGGATATTTATA-3′) of Flk-1 or the vector with non-sense oligonucleotides as a control was transfected into U87 cells using FuGENE 6.

    Techniques: shRNA, Expressing, Plasmid Preparation, Injection, Mouse Assay

    EBNA3C blocks p73 and Apaf-1 expressions by inhibiting the DNA-binding ability of E2F1 to its targeted promoters in LCLs. A–B) Approximately10 million human peripheral blood mononuclear cells (PBMC) were infected by either A) wild-type (WT) BAC GFP-EBV or EBNA3C knockout BAC GFP-EBV (ΔE3C) for 4 h. At indicates times post-infection cells were harvested, total RNA was isolated and subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1 and EBNA3C transcript levels. Each sample was tested in triplicate and data obtained from two independent experiments with two different donors and expressed as the difference of the quantity of specific transcripts to the quantity of GAPDH transcript as control. The fold change in expression of each mRNA relative to GAPDH transcript was calculated based on the threshold cycle (Ct) as 2 − Δ(ΔCt) , where ΔCt = Ct target −Ct GAPDH and Δ(ΔCt) = ΔCt test sample −ΔCt control sample . C) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins were subjected to western blot (WB) analysis using indicated antibodies. D) Approximately 20 million of EBNA3C knockdown LCLs were transfected with 50 ìg of plasmids expressing either vector control or myc-tagged EBNA3C via electroporation. Transfected LCLs were cultured in RPMI medium with 10% FBS for 48 h and subjected for western blot analysis using indicated antibodies. E) Total RNA was isolated from indicated LCLs, subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1, p73 and Apaf-1 transcript levels as similar to A–B). F) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins (50 µg) were subjected to western blot analysis using indicated antibodies. G) A ChIP assay was performed using either control or EBNA3C knockdown LCLs. Material immunoprecipitated with anti-E2F1 or control antibody (rabbit IgG) was amplified by using primers specific for p73 (top) or Apaf1 (bottom) promoters. The end products of qPCR bands ran into a 2.5% agarose gel. Bar diagrams represent the change in Ct value (ΔCt) over IgG. H–I) Saos-2 (p53 −/− pRb −/− ) cells were transfected with either 5 µg of the wild-type H) p73-luciferase or I) Apaf-1-luciferase reporter plasmids with flag-E2F1 and myc-EBNA3C expression vectors as indicated. Luciferase activity was assessed at 36 h of post-transfection. J) Schematic representation of streptavidin pulldown assay as described in ‘ Materials and Methods ’ section. K) 100 µg of cell extracts from Saos-2 cells transfected with flag-E2F1 with or without myc-EBNA3C expression vector were incubated with 200 ng of the indicated biotinylated oligonucleotides (WT or Mut) immobilized with streptavidin accordingly to the manufacturer protocol, in the absence or presence of a 200 molar excess of the corresponding non biotinylated oligonucleotide (competition: comp). Oligonucleotide-bound E2F1 protein was detected by western blotting using anti-flag antibody (M2). The binding capacity of each oligonucleotide is given as percentage at bottom. All panels are representative of two independent experiments.

    Journal: PLoS Pathogens

    Article Title: E2F1 Mediated Apoptosis Induced by the DNA Damage Response Is Blocked by EBV Nuclear Antigen 3C in Lymphoblastoid Cells

    doi: 10.1371/journal.ppat.1002573

    Figure Lengend Snippet: EBNA3C blocks p73 and Apaf-1 expressions by inhibiting the DNA-binding ability of E2F1 to its targeted promoters in LCLs. A–B) Approximately10 million human peripheral blood mononuclear cells (PBMC) were infected by either A) wild-type (WT) BAC GFP-EBV or EBNA3C knockout BAC GFP-EBV (ΔE3C) for 4 h. At indicates times post-infection cells were harvested, total RNA was isolated and subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1 and EBNA3C transcript levels. Each sample was tested in triplicate and data obtained from two independent experiments with two different donors and expressed as the difference of the quantity of specific transcripts to the quantity of GAPDH transcript as control. The fold change in expression of each mRNA relative to GAPDH transcript was calculated based on the threshold cycle (Ct) as 2 − Δ(ΔCt) , where ΔCt = Ct target −Ct GAPDH and Δ(ΔCt) = ΔCt test sample −ΔCt control sample . C) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins were subjected to western blot (WB) analysis using indicated antibodies. D) Approximately 20 million of EBNA3C knockdown LCLs were transfected with 50 ìg of plasmids expressing either vector control or myc-tagged EBNA3C via electroporation. Transfected LCLs were cultured in RPMI medium with 10% FBS for 48 h and subjected for western blot analysis using indicated antibodies. E) Total RNA was isolated from indicated LCLs, subjected to cDNA preparation as per manufacturer's instruction followed by quantitative real-time PCR analysis for detecting E2F1, p73 and Apaf-1 transcript levels as similar to A–B). F) Approximately 10 million of EBNA3C and control knockdown LCLs were harvested and total cell proteins (50 µg) were subjected to western blot analysis using indicated antibodies. G) A ChIP assay was performed using either control or EBNA3C knockdown LCLs. Material immunoprecipitated with anti-E2F1 or control antibody (rabbit IgG) was amplified by using primers specific for p73 (top) or Apaf1 (bottom) promoters. The end products of qPCR bands ran into a 2.5% agarose gel. Bar diagrams represent the change in Ct value (ΔCt) over IgG. H–I) Saos-2 (p53 −/− pRb −/− ) cells were transfected with either 5 µg of the wild-type H) p73-luciferase or I) Apaf-1-luciferase reporter plasmids with flag-E2F1 and myc-EBNA3C expression vectors as indicated. Luciferase activity was assessed at 36 h of post-transfection. J) Schematic representation of streptavidin pulldown assay as described in ‘ Materials and Methods ’ section. K) 100 µg of cell extracts from Saos-2 cells transfected with flag-E2F1 with or without myc-EBNA3C expression vector were incubated with 200 ng of the indicated biotinylated oligonucleotides (WT or Mut) immobilized with streptavidin accordingly to the manufacturer protocol, in the absence or presence of a 200 molar excess of the corresponding non biotinylated oligonucleotide (competition: comp). Oligonucleotide-bound E2F1 protein was detected by western blotting using anti-flag antibody (M2). The binding capacity of each oligonucleotide is given as percentage at bottom. All panels are representative of two independent experiments.

    Article Snippet: Cells were additionally transfected with a GFP expression vector (pEGFP-C1, BD Biosciences Clontech).

    Techniques: Binding Assay, Infection, BAC Assay, Knock-Out, Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Plasmid Preparation, Electroporation, Cell Culture, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Luciferase, Activity Assay, Incubation

    EBNA3C expression leads to E2F1 destabilization via an ubiquitin-proteasome dependent pathway. A) HEK 293 cells were co-transfected with flag-E2F1 and either vector control (lanes 1 and 3) or myc-EBNA3C (lanes 2 and 4) with GFP expressing vector. At 36 h posttransfection, samples were treated with either 20 µM MG132 (+lanes) or DMSO (−lanes) for 6 h and resolved by 10% SDS-PAGE and probed with the indicated antibodies. B) HEK 293 cells were similarly transfected with expression plasmids for flag-E2F1, myc-tagged EBNA3C and GFP plasmids as indicated. At 36 h post-transfection, cells were treated with 40 µg/ml cyclohexamide (CHX) for indicated lengths of time. 5% of the lysate from each sample were resolved by 9% SDS-PAGE and western blotted with indicated antibodies. C) 15 million HEK 293 cells transfected with different combinations of expression plasmids as indicated. Cells were harvested at 36 h, and total protein was immunoprecipitated (IP) with flag antibody and samples were resolved by 9% SDS-PAGE. D) Approximately 10 million of stably generated LCLs with either Sh-control (Sh-Con) or Sh-EBNA3C (Sh-E3C) were incubated with 100 µg/ml CHX for indicated lengths of time in RPMI medium containing either 10% FBS (+serum/DMSO) or 0.1% FBS plus 5 µM etoposide (−serum/+Etop) at 37°C. 10% of the lysate from each sample were resolved by 9% SDS-PAGE and western blotted with indicated antibodies. E) Approximately 50 million LCLs with either control Sh-RNA (Sh-Con) or EBNA3C directed Sh-RNA (Sh-E3C) were harvested after 10 h incubation with proteasome inhibitor MG132 (40 µM). Cells were lysed and E2F1 was immunoprecipitated (IP). Samples were resolved by 9% SDS-PAGE and western blotting (WB) was done by stripping and reprobing the same membrane. F) Saos-2 cells transfected with the WT E2F1 reporter plasmid in the presence of either flag-E2F1 alone or flag-E2F1 plus myc-EBNA3C expressing plasmids followed by incubated with either DMSO or MG132 (20 µM) were subjected for reporter assay as essentially described in ‘ Materials and Methods ’. Mean values and standard deviations of three independent experiments are presented. Bottoms panels indicate a representative blot of 5% of the total cell lysates resolved by appropriate % SDS-PAGE demonstrating the expression levels of ectopically expressed proteins. E2F1 protein bands were quantified using Odyssey imager software as indicated as either bar diagrams (A, B and D) or arbitrary numerical values (relative intensity, RI) at the bottom of gel (B–E) based on GFP or GAPDH loading controls, where applicable. G–H) Approximately 10 million of indicated cells incubated with either DMSO or MG132 (40 µM) for 10 h, harvested and 10% of total lysates were subjected for western blots with indicated antibodies. For all western blots, where appropriate, GAPDH serves as an internal loading control and GFP as a transfection efficiency control. Western blotting was done by stripping and reprobing the same membrane. Protein bands were quantified using Odyssey imager software as indicated either as arbitrary numerical values at the bottom of gel or as bar diagrams based on either GAPDH or GFP loading control. I) In response to DNA damage signals E2F1 gets stabilized and transcriptionally activates pro-apoptotic genes p73 and Apaf-1, which eventually induces apoptosis. In EBV transformed cells, by forming a stable complex with E2F1, EBNA3C inhibits its DNA binding activity and inhibits the transcriptional activation of p73 and Apaf-1. Moreover, EBNA3C specifically targets E2F1 for an ubiquitin-proteasome mediated degradation, which altogether blocks apoptotic induction. Moreover, sh-RNA designed against E2F1 showed reverse consequences and augments the apoptotic resistance of the cells.

    Journal: PLoS Pathogens

    Article Title: E2F1 Mediated Apoptosis Induced by the DNA Damage Response Is Blocked by EBV Nuclear Antigen 3C in Lymphoblastoid Cells

    doi: 10.1371/journal.ppat.1002573

    Figure Lengend Snippet: EBNA3C expression leads to E2F1 destabilization via an ubiquitin-proteasome dependent pathway. A) HEK 293 cells were co-transfected with flag-E2F1 and either vector control (lanes 1 and 3) or myc-EBNA3C (lanes 2 and 4) with GFP expressing vector. At 36 h posttransfection, samples were treated with either 20 µM MG132 (+lanes) or DMSO (−lanes) for 6 h and resolved by 10% SDS-PAGE and probed with the indicated antibodies. B) HEK 293 cells were similarly transfected with expression plasmids for flag-E2F1, myc-tagged EBNA3C and GFP plasmids as indicated. At 36 h post-transfection, cells were treated with 40 µg/ml cyclohexamide (CHX) for indicated lengths of time. 5% of the lysate from each sample were resolved by 9% SDS-PAGE and western blotted with indicated antibodies. C) 15 million HEK 293 cells transfected with different combinations of expression plasmids as indicated. Cells were harvested at 36 h, and total protein was immunoprecipitated (IP) with flag antibody and samples were resolved by 9% SDS-PAGE. D) Approximately 10 million of stably generated LCLs with either Sh-control (Sh-Con) or Sh-EBNA3C (Sh-E3C) were incubated with 100 µg/ml CHX for indicated lengths of time in RPMI medium containing either 10% FBS (+serum/DMSO) or 0.1% FBS plus 5 µM etoposide (−serum/+Etop) at 37°C. 10% of the lysate from each sample were resolved by 9% SDS-PAGE and western blotted with indicated antibodies. E) Approximately 50 million LCLs with either control Sh-RNA (Sh-Con) or EBNA3C directed Sh-RNA (Sh-E3C) were harvested after 10 h incubation with proteasome inhibitor MG132 (40 µM). Cells were lysed and E2F1 was immunoprecipitated (IP). Samples were resolved by 9% SDS-PAGE and western blotting (WB) was done by stripping and reprobing the same membrane. F) Saos-2 cells transfected with the WT E2F1 reporter plasmid in the presence of either flag-E2F1 alone or flag-E2F1 plus myc-EBNA3C expressing plasmids followed by incubated with either DMSO or MG132 (20 µM) were subjected for reporter assay as essentially described in ‘ Materials and Methods ’. Mean values and standard deviations of three independent experiments are presented. Bottoms panels indicate a representative blot of 5% of the total cell lysates resolved by appropriate % SDS-PAGE demonstrating the expression levels of ectopically expressed proteins. E2F1 protein bands were quantified using Odyssey imager software as indicated as either bar diagrams (A, B and D) or arbitrary numerical values (relative intensity, RI) at the bottom of gel (B–E) based on GFP or GAPDH loading controls, where applicable. G–H) Approximately 10 million of indicated cells incubated with either DMSO or MG132 (40 µM) for 10 h, harvested and 10% of total lysates were subjected for western blots with indicated antibodies. For all western blots, where appropriate, GAPDH serves as an internal loading control and GFP as a transfection efficiency control. Western blotting was done by stripping and reprobing the same membrane. Protein bands were quantified using Odyssey imager software as indicated either as arbitrary numerical values at the bottom of gel or as bar diagrams based on either GAPDH or GFP loading control. I) In response to DNA damage signals E2F1 gets stabilized and transcriptionally activates pro-apoptotic genes p73 and Apaf-1, which eventually induces apoptosis. In EBV transformed cells, by forming a stable complex with E2F1, EBNA3C inhibits its DNA binding activity and inhibits the transcriptional activation of p73 and Apaf-1. Moreover, EBNA3C specifically targets E2F1 for an ubiquitin-proteasome mediated degradation, which altogether blocks apoptotic induction. Moreover, sh-RNA designed against E2F1 showed reverse consequences and augments the apoptotic resistance of the cells.

    Article Snippet: Cells were additionally transfected with a GFP expression vector (pEGFP-C1, BD Biosciences Clontech).

    Techniques: Expressing, Transfection, Plasmid Preparation, SDS Page, Western Blot, Immunoprecipitation, Stable Transfection, Generated, Incubation, Stripping Membranes, Reporter Assay, Software, Transformation Assay, Binding Assay, Activity Assay, Activation Assay

    Co-expression of EBNA3C blocks E2F1 mediated transcriptional activity. A) The schematic represents three wild-type (WT) and three mutant (Mut) copies of the E2F1 responsive promoter element fused with the luciferase gene in pGL2Basic. B) HEK 293 (pRb +/+ ) cells were co-transfected with either 10 µg of WT (blue) or Mut (red) E2F1 reporter plasmids in combinations of expression plasmids for myc-EBNA3C and flag-E2F1 as indicated. C–E) Saos-2 (pRb −/− ) cells were transfected with WT E2F1 reporter plasmid in the presence of different expression constructs as indicated. Cells were additionally transfected with 5 µg of pCMV-βgal and pEGFP-C1 expression vectors to normalize transfection efficiency. At 36 h post-transfection, cells were harvested and lysed in reporter lysis buffer. Total amount of proteins were normalized by Bradford assay and both luciferase and β-galactosidase activities were measured as described in ‘ Materials and Methods ’ section. Mean values and standard deviations of three independent experiments are presented. Bottoms panels indicate a representative blot of 5% of the total cell lysates resolved by appropriate % SDS-PAGE demonstrating the expression levels of ectopically expressed proteins. GAPDH blot was done as an internal loading control. E2F1 protein bands were quantified using Odyssey imager software as indicated as arbitrary numerical values (relative intensity, RI) at the bottom of gel (B–E) based on both GFP and GAPDH loading controls. F) HEK293 cells transfected with the WT E2F1 reporter plasmid in the presence of either flag-E2F1 alone or flag-E2F1 plus myc-EBNA3C expressing plasmids were subjected to ChIP assay as described in ‘ Materials and Methods ’ section. The eluted DNA samples were subjected to qPCR analysis using primers directed for either E2F1-responsive promoter fused with luciferase gene (top) or SV-40 promoter region (bottom). Panels show representative pictures from two independent experiments.

    Journal: PLoS Pathogens

    Article Title: E2F1 Mediated Apoptosis Induced by the DNA Damage Response Is Blocked by EBV Nuclear Antigen 3C in Lymphoblastoid Cells

    doi: 10.1371/journal.ppat.1002573

    Figure Lengend Snippet: Co-expression of EBNA3C blocks E2F1 mediated transcriptional activity. A) The schematic represents three wild-type (WT) and three mutant (Mut) copies of the E2F1 responsive promoter element fused with the luciferase gene in pGL2Basic. B) HEK 293 (pRb +/+ ) cells were co-transfected with either 10 µg of WT (blue) or Mut (red) E2F1 reporter plasmids in combinations of expression plasmids for myc-EBNA3C and flag-E2F1 as indicated. C–E) Saos-2 (pRb −/− ) cells were transfected with WT E2F1 reporter plasmid in the presence of different expression constructs as indicated. Cells were additionally transfected with 5 µg of pCMV-βgal and pEGFP-C1 expression vectors to normalize transfection efficiency. At 36 h post-transfection, cells were harvested and lysed in reporter lysis buffer. Total amount of proteins were normalized by Bradford assay and both luciferase and β-galactosidase activities were measured as described in ‘ Materials and Methods ’ section. Mean values and standard deviations of three independent experiments are presented. Bottoms panels indicate a representative blot of 5% of the total cell lysates resolved by appropriate % SDS-PAGE demonstrating the expression levels of ectopically expressed proteins. GAPDH blot was done as an internal loading control. E2F1 protein bands were quantified using Odyssey imager software as indicated as arbitrary numerical values (relative intensity, RI) at the bottom of gel (B–E) based on both GFP and GAPDH loading controls. F) HEK293 cells transfected with the WT E2F1 reporter plasmid in the presence of either flag-E2F1 alone or flag-E2F1 plus myc-EBNA3C expressing plasmids were subjected to ChIP assay as described in ‘ Materials and Methods ’ section. The eluted DNA samples were subjected to qPCR analysis using primers directed for either E2F1-responsive promoter fused with luciferase gene (top) or SV-40 promoter region (bottom). Panels show representative pictures from two independent experiments.

    Article Snippet: Cells were additionally transfected with a GFP expression vector (pEGFP-C1, BD Biosciences Clontech).

    Techniques: Expressing, Activity Assay, Mutagenesis, Luciferase, Transfection, Plasmid Preparation, Construct, Lysis, Bradford Assay, SDS Page, Software, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    EBNA3C forms a pRb independent complex with E2F1. 50 million A) DG75 and two LCL clones (LCL1 and 2) and B) BJAB and two BJAB stable clones expressing EBNA3C (E3C7 and E3C10) were subjected to immunoprecipitation (IP) with EBNA3C specific rabbit antibody. Samples were resolved by SDS-PAGE and detected by western blot (WB) for the indicated proteins by stripping and reprobing the same membrane. 10 million C) HEK293 (pRb +/+ ) or D) Saos-2 (pRb −/− ) cells were co-transfected with plasmids expressing myc-EBNA3C either in the presence of vector control, wild-type flag-E2F1 (residues 1–437) or pRb binding deficient flag-E2F1 (residues 1–400) as indicated. At 36 h post-transfection, cells were harvested, lysed in RIPA buffer and IP with flag-antibody. Samples were western blotted (WB) with the indicated antibodies. The asterisks indicate the immunoglobulin bands. E) EBV transformed cells LCL2 were plated and air-dried onto slides. F) Saos-2 (pRb −/− ) cells plated on coverslips were co-transfected with plasmids expressing GFP-EBNA3C with flag-E2F1 using Lipofectamine 2000 as per manufactures instructions. E) Endogenously and F) ectopically expressed E2F1 were detected by either specific rabbit antibody (C-20) or mouse M2-antibody, respectively, followed by specific anti-Alexa Fluor 594 2 0 antibody (red). A) Endogenous EBNA3C in EBV positive LCLs was detected using an EBNA3C-specific antibody (A10 ascites) followed by 2 0 antibody anti-mouse Alexa Fluor 488 (green). Ectopically expressed GFP-EBNA3C in Saos-2 cells was detected by GFP fluorescence. The nuclei were subsequently stained with DAPI and the images were captured using an Olympus confocal microscope. All panels are representative pictures from approximately 100 cells of 10 different fields of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: E2F1 Mediated Apoptosis Induced by the DNA Damage Response Is Blocked by EBV Nuclear Antigen 3C in Lymphoblastoid Cells

    doi: 10.1371/journal.ppat.1002573

    Figure Lengend Snippet: EBNA3C forms a pRb independent complex with E2F1. 50 million A) DG75 and two LCL clones (LCL1 and 2) and B) BJAB and two BJAB stable clones expressing EBNA3C (E3C7 and E3C10) were subjected to immunoprecipitation (IP) with EBNA3C specific rabbit antibody. Samples were resolved by SDS-PAGE and detected by western blot (WB) for the indicated proteins by stripping and reprobing the same membrane. 10 million C) HEK293 (pRb +/+ ) or D) Saos-2 (pRb −/− ) cells were co-transfected with plasmids expressing myc-EBNA3C either in the presence of vector control, wild-type flag-E2F1 (residues 1–437) or pRb binding deficient flag-E2F1 (residues 1–400) as indicated. At 36 h post-transfection, cells were harvested, lysed in RIPA buffer and IP with flag-antibody. Samples were western blotted (WB) with the indicated antibodies. The asterisks indicate the immunoglobulin bands. E) EBV transformed cells LCL2 were plated and air-dried onto slides. F) Saos-2 (pRb −/− ) cells plated on coverslips were co-transfected with plasmids expressing GFP-EBNA3C with flag-E2F1 using Lipofectamine 2000 as per manufactures instructions. E) Endogenously and F) ectopically expressed E2F1 were detected by either specific rabbit antibody (C-20) or mouse M2-antibody, respectively, followed by specific anti-Alexa Fluor 594 2 0 antibody (red). A) Endogenous EBNA3C in EBV positive LCLs was detected using an EBNA3C-specific antibody (A10 ascites) followed by 2 0 antibody anti-mouse Alexa Fluor 488 (green). Ectopically expressed GFP-EBNA3C in Saos-2 cells was detected by GFP fluorescence. The nuclei were subsequently stained with DAPI and the images were captured using an Olympus confocal microscope. All panels are representative pictures from approximately 100 cells of 10 different fields of three independent experiments.

    Article Snippet: Cells were additionally transfected with a GFP expression vector (pEGFP-C1, BD Biosciences Clontech).

    Techniques: Clone Assay, Expressing, Immunoprecipitation, SDS Page, Western Blot, Stripping Membranes, Transfection, Plasmid Preparation, Binding Assay, Transformation Assay, Fluorescence, Staining, Microscopy

    EBNA3C inhibits E2F1 mediated anti-proliferative activities in Saos-2 (p53 −/− pRb −/− ) cells. A–D) Saos-2 (pRb −/− ) cells were electroporated with expression plasmids for either empty vector, flag-E2F1, myc-EBNA3C or myc-EBNA3C residues 366–620 as indicated. A–C) Cells were additionally transfected with a GFP expression vector. At 24 h post-transfection cells were exposed to serum starvation and 5 µM etoposide treatment for 12 h, followed by selection with G418 for 2 weeks. A) After a 2-week selection, cells were fixed on the plates with 4% formaldehyde and scanned for GFP expressed colonies using Typhoon 9410 imaging system. The area of stained cells in each dish was calculated by Image J software. B) Approximately 0.1×10 6 of flag-E2F1 and flag-E2F1 plus either full-length myc-EBNA3C or myc-EBNA3C residues 366–620 expressing selected cells from each set of samples were plated into each well of the 6-well plates and cultured for 6 days after 12 h treatment with serum starvation and 5 µM etoposide. Viable cells from each well were counted by trypan blue exclusion method daily using an automated cell counter. C) Selected cells with similar treatment as A) were subjected to flow cytometry analyses as described in ‘ Materials and Methods ’ section. Bar diagrams represent average sub G1 values of two independent experiments. D) Saos-2 cells transfected with different combinations of expression plasmids as described in panel A) and selected similarly as stated above with G418. After genotoxic stress with serum starvation and 5 µM etoposide treatment for 12 h cells were fixed and subjected for TUNEL assay as per manufactures protocol. A–D) All panels are representative of two independent experiments and bar diagrams represent the average data of two independent experiments with standard deviation.

    Journal: PLoS Pathogens

    Article Title: E2F1 Mediated Apoptosis Induced by the DNA Damage Response Is Blocked by EBV Nuclear Antigen 3C in Lymphoblastoid Cells

    doi: 10.1371/journal.ppat.1002573

    Figure Lengend Snippet: EBNA3C inhibits E2F1 mediated anti-proliferative activities in Saos-2 (p53 −/− pRb −/− ) cells. A–D) Saos-2 (pRb −/− ) cells were electroporated with expression plasmids for either empty vector, flag-E2F1, myc-EBNA3C or myc-EBNA3C residues 366–620 as indicated. A–C) Cells were additionally transfected with a GFP expression vector. At 24 h post-transfection cells were exposed to serum starvation and 5 µM etoposide treatment for 12 h, followed by selection with G418 for 2 weeks. A) After a 2-week selection, cells were fixed on the plates with 4% formaldehyde and scanned for GFP expressed colonies using Typhoon 9410 imaging system. The area of stained cells in each dish was calculated by Image J software. B) Approximately 0.1×10 6 of flag-E2F1 and flag-E2F1 plus either full-length myc-EBNA3C or myc-EBNA3C residues 366–620 expressing selected cells from each set of samples were plated into each well of the 6-well plates and cultured for 6 days after 12 h treatment with serum starvation and 5 µM etoposide. Viable cells from each well were counted by trypan blue exclusion method daily using an automated cell counter. C) Selected cells with similar treatment as A) were subjected to flow cytometry analyses as described in ‘ Materials and Methods ’ section. Bar diagrams represent average sub G1 values of two independent experiments. D) Saos-2 cells transfected with different combinations of expression plasmids as described in panel A) and selected similarly as stated above with G418. After genotoxic stress with serum starvation and 5 µM etoposide treatment for 12 h cells were fixed and subjected for TUNEL assay as per manufactures protocol. A–D) All panels are representative of two independent experiments and bar diagrams represent the average data of two independent experiments with standard deviation.

    Article Snippet: Cells were additionally transfected with a GFP expression vector (pEGFP-C1, BD Biosciences Clontech).

    Techniques: Expressing, Plasmid Preparation, Transfection, Selection, Imaging, Staining, Software, Cell Culture, Flow Cytometry, Cytometry, TUNEL Assay, Standard Deviation