pcr4 expression vector Thermo Fisher Search Results


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  • 99
    Thermo Fisher pcr4 cloning vector
    Pcr4 Cloning Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr4 expression vector
    Pcr4 Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr4 vector
    Pcr4 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr4 topo vector
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to <t>pCR4‐TOPO</t> vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Pcr4 Topo Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 8236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher intermediate vector pcr4 topo ta
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to <t>pCR4‐TOPO</t> vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Intermediate Vector Pcr4 Topo Ta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr4 blunt topo vector
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to <t>pCR4‐TOPO</t> vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Pcr4 Blunt Topo Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 471 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr4 topo sequencing vector
    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to <t>pCR4‐TOPO</t> vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).
    Pcr4 Topo Sequencing Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna4 to expression vectors
    C/EBP-β LIP induces calreticulin expression and immunogenic cell death. MDA-MB-231 cells were left untreated (−, ctrl) or transfected with an empty <t>pcDNA4/TO</t> vector (em/empty), with a pcDNA4/TO expression vector encoding C/EBP-β LAP or C/EBP-β LIP, respectively. a. Whole cell lysates were probed with an antibody recognizing both C/EBP-β LAP and LIP isoforms. The expression of β-tubulin was used as control of equal protein loading. The figure is representative of 1 out of 3 experiments. b. ChIP was performed to evaluate the binding of LAP or LIP to the CRT promoter (sites: 831–843; 1302–1313). no Ab: no anti-C/EBP-β antibody; bl: blank; DNA input: genomic DNA. The figure is representative of 1 out of 3 experiments. c. The relative expression of CRT gene was measured in triplicates by qRT-PCR. Data are presented as means±SD (n = 3). * p
    Pcdna4 To Expression Vectors, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher clonejet pcr cloning kit
    C/EBP-β LIP induces calreticulin expression and immunogenic cell death. MDA-MB-231 cells were left untreated (−, ctrl) or transfected with an empty <t>pcDNA4/TO</t> vector (em/empty), with a pcDNA4/TO expression vector encoding C/EBP-β LAP or C/EBP-β LIP, respectively. a. Whole cell lysates were probed with an antibody recognizing both C/EBP-β LAP and LIP isoforms. The expression of β-tubulin was used as control of equal protein loading. The figure is representative of 1 out of 3 experiments. b. ChIP was performed to evaluate the binding of LAP or LIP to the CRT promoter (sites: 831–843; 1302–1313). no Ab: no anti-C/EBP-β antibody; bl: blank; DNA input: genomic DNA. The figure is representative of 1 out of 3 experiments. c. The relative expression of CRT gene was measured in triplicates by qRT-PCR. Data are presented as means±SD (n = 3). * p
    Clonejet Pcr Cloning Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcr2 1 ta cloning vector
    DJH1 joint sequences from MSH2 +/+ and MSH2 −/− littermates. DJH PCR products from the reaction shown in Figure 1B were used as templates in a nested PCR using a JH1–JH2 intronic primer (J1CY) to amplify only joints that utilized the JH1 segment. PCR products were cloned into the <t>PCR2.1</t> vector and sequenced. ( A ) Upper panel shows sequences from littermate WT-1 and lower panel shows sequences from littermate WT-2. ( B ) Upper panel shows sequences from littermate MSH2 −/− 1 and lower panel shows sequences from littermate MSH2 −/− 2. Sequences followed by an asterisk were scored as joining by homology joints. P-nucleotides are indicated in bold.
    Pcr2 1 Ta Cloning Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 955 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher alicator lic cloning
    DJH1 joint sequences from MSH2 +/+ and MSH2 −/− littermates. DJH PCR products from the reaction shown in Figure 1B were used as templates in a nested PCR using a JH1–JH2 intronic primer (J1CY) to amplify only joints that utilized the JH1 segment. PCR products were cloned into the <t>PCR2.1</t> vector and sequenced. ( A ) Upper panel shows sequences from littermate WT-1 and lower panel shows sequences from littermate WT-2. ( B ) Upper panel shows sequences from littermate MSH2 −/− 1 and lower panel shows sequences from littermate MSH2 −/− 2. Sequences followed by an asterisk were scored as joining by homology joints. P-nucleotides are indicated in bold.
    Alicator Lic Cloning, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pproex htb expression plasmid
    Purified rMG309c induced proinflammatory cytokine elaboration. (A) The largest coding fragment of MG309 (designated rMG309c) free of UGA codons (stop signal [St] in E. coli ) was PCR amplified and cloned into <t>pPROEX-HTb</t> for direct expression of a six-His-tagged
    Pproex Htb Expression Plasmid, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pcdna4 vector
    The level of anti-SjGST in serum samples after vaccination and after Schistosoma japonicum challenge. Serum samples were collected from the tails of mice immunized with <t>pcDNA4</t> (white), pcDNA/SjGST (light grey), pcDNA/SjGST + pIL-12 (dark grey), pcDNA/SjGST + rSjGST (black), or pcDNA/SjGST + pIL-12 + rSjGST (mosaic), 8 weeks after vaccination (left panel) or 8 weeks after infection (right panel). The antibody titers of (A) total IgG, (B) IgG1, (C) IgG2a, and (D) IgG2b were measured by ELISA. The OD405 value of each group was directly shown in the figure. The results are shown as the mean ± standard deviation from three independent experiments. *: p
    Pcdna4 Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher expression vector ppro ex htb
    The level of anti-SjGST in serum samples after vaccination and after Schistosoma japonicum challenge. Serum samples were collected from the tails of mice immunized with <t>pcDNA4</t> (white), pcDNA/SjGST (light grey), pcDNA/SjGST + pIL-12 (dark grey), pcDNA/SjGST + rSjGST (black), or pcDNA/SjGST + pIL-12 + rSjGST (mosaic), 8 weeks after vaccination (left panel) or 8 weeks after infection (right panel). The antibody titers of (A) total IgG, (B) IgG1, (C) IgG2a, and (D) IgG2b were measured by ELISA. The OD405 value of each group was directly shown in the figure. The results are shown as the mean ± standard deviation from three independent experiments. *: p
    Expression Vector Ppro Ex Htb, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher psilencer 4 1 cmv puro mammalian expression vector
    miR-199a-3p suppressed RA-FLS proliferation ( A ) RT-qPCR of miR-199a-3p in RA-FLSs transfected with pSilencer 4.1-miR-199a-3p plasmid (miR-199a-3p) or no-insert control pSilencer <t>4.1-CMV</t> <t>puro</t> plasmid (Mock). ( B ) Forty-eight hours post transfection, MTT assay was performed to test the viability of miR-199a-3p or Mock RA-FLSs. ( C , D ). EdU assay of miR-199a-3p or Mock transfected RA-FLSs 48 h post transfection. Cells were stained for EdU and Hoechst (to mark nuclei) (C), and quantitated for EdU+ cell percentage (D). CCK8 assay showed that ectopic expression of miR-199a-3p significantly inhibited RA-FLS proliferation rate over 5 days. n =4 or n =6 (A,D); * P
    Psilencer 4 1 Cmv Puro Mammalian Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna
    miR390 Expression Responds to Auxin during Lateral Root Induction. (A) <t>RNA</t> gel blot analysis of 15 μg of root RNA from 10-d-old wild-type plants during a time course of 10 μM auxin (IAA) treatment after 24 h of 10 μM NPA pretreatment. The blot was successively probed with DNA complementary to miR390, miR156, and miR160. U6 snRNA served as a loading control. (B) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type plants. Plants were pretreated with 10 μM NPA and then for another 24 h with either DMSO (–), 10 μM cycloheximide (CHX), 10 μM IAA, or both (CHX+IAA). U6 snRNA served as a loading control. (C) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type or mir390a plants treated (+) or untreated (−) for 24 h with 10 μM IAA after 24 h of 10 μM NPA pretreatment. In (A) to (C) , numbers are the ratios of miR390 to U6 signal. These experiments were done twice with similar results. (D) <t>RT-PCR</t> analysis of root RNA from 10-d-old wild-type plants treated for 24 h with 10 μM IAA (+) or untreated (−) after NPA pretreatment. Primers amplify specifically the MIR390a precursor and ACTIN2 (loading control) from cDNA (RT+) but not from genomic DNA (RT−). (E) and (F) Confocal analysis of pMIR390a:GUS-GFP expression in 10-d-old wild-type plants treated (+IAA) or untreated (Control) for 10 h with 10 μM IAA after NPA pretreatment. GFP is in green, and cell walls are stained by propidium iodide in red. Bars = 50 μm.
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 177224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ebv mirna expression vector
    <t>EBV</t> miR-BHRF1-2-5p, miR-BART2-5p, and cellular miR-17 regulate the latent to lytic switch. A and C-G. MutuI cells were transduced with pLCE control vector or sponge inhibitors to indicated EBV or cellular miRNAs, then treated for 24 or 42 hrs with 5 ug/mL anti-IgM. Total RNA was harvested and assayed by qRT-PCR for EBV gene expression as indicated. Reported are the averages of two independent experiments with qPCR performed in duplicate; expression levels are normalized to GAPDH and shown relative to mock treated cells (harvested at 42 hrs) for each <t>miRNA</t> sponge inhibitor. B. miRNA levels in anti-IgM treated, sponged MutuI cells assayed by qRT-PCR. Values are normalized to miR-16 and shown relative to pLCE control cells for each respective sponge inhibitor. Reported are the averages of two independent experiments with qPCR performed in duplicate. *By Student’s t test, p
    Ebv Mirna Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lentiviral vectors encoding shrna against pdcd4
    <t>PDCD4</t> as negative regulator of dermal fibroblast senescence and CAF activation A. Clonogenecity assays. HDFs were infected with two <t>shRNA</t> PDCD4-silencing lentiviruses in parallel with empty vector control, followed by selection with puromycin for 48hrs, then plated under low density conditions on triplicated dishes (1000 cells per 60mm dish). Colony formation was measured by crystal violet staining after 14 days. B. Senescence Associated β-galactosidase activity (SA β-gal) assay. HDFs infected with the PDCD4-silencing and control lentiviruses as in the previous panel were stained with SA β-gal at 7 days after infection. Representative images (left panel) and quantification of positive SA β-gal staining in ∼100 cells (right panel) are shown. C. Parallel cultures of cells as in the previous panel were analyzed for expression of PDCD4 gene silencing and expression of the senescence-determinant CDKN1A gene by RT-PCR. D. , E. HDFs were transfected with two different PDCD4-silencing siRNAs in parallel with scrambled siRNA control, followed, 72hrs after transfection, by analysis of expression of the indicated genes by real time RT-PCR. Insert: Immunoblot analysis of HDFs plus/minus siRNA-mediated silencing of PDCD4. F. HDFs, Saos-2 cells, human primary keratinocytes (HKCs) and HeLa cells were transfected with siRNAs for silencing of the CSL or PDCD4 genes in parallel with scrambled siRNA controls, followed by determination of HES1 expression by real time RT-PCR. G. , H. Immunoblot analysis of CSL and PDCD4 expression in several HDF and HKC strains and Saos-2 and HeLa cells as indicated, with tubulin as equal loading control.
    Lentiviral Vectors Encoding Shrna Against Pdcd4, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Journal: Clinical and Experimental Immunology

    Article Title: Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer

    doi: 10.1111/cei.13183

    Figure Lengend Snippet: DNA methylation in the enhancer region regulates perforin transcription. (a) Promoter region sequence containing enhancer in the PRF1 gene was aligned between human and mouse using the VISTA tool. The conservation degree is indicated by the peaks, with coloured peaks (blue for exon and light red for the non‐coding sequence) indicating > 70% conservation. Ten cytosine–phosphate–guanine sites (CpGs) in the enhancer are displayed upstream from the transcription start site (TSS). (b) Sorted CD8 + T cells from healthy donors were cultured initially in the presence of 200 IU/ml recombinant human interleukin (IL)‐2, 5 μg/ml plate‐coated anti‐CD3 and 1 μg/ml anti‐CD28 stimulating antibodies in vitro . 5‐Azacytidine (5‐aza) was added to the culture at a final concentration of 2·5 μM/ml for 48 h, followed by culture medium replacement and incubation for another 48 h. Flow cytometry data demonstrating perforin expression pre‐ and post‐treatment are shown. (c) Perforin – and perforin + CD8 + T cells were sorted. DNA was extracted and bisulfite‐converted from each cell subset, TA‐cloned to pCR4‐TOPO vector and transformed into TOP10 Escherichia coli . Colonies were picked from each cell subset and their DNA was sequenced to measure DNA methylation status of the 10 CpGs in the enhancer. The data display rows represent individually sequenced clone. (d) The bar graphs display total DNA methylation percentage in each CpG from clones in (c).

    Article Snippet: Next, the PCR amplicons were TA‐cloned into pCR4‐TOPO vector (ThermoFisher) and transformed into TOP10 Escherichia coli (ThermoFisher) by heat shock, according to the manufacturer’s protocol.

    Techniques: DNA Methylation Assay, Sequencing, Cell Culture, Recombinant, In Vitro, Concentration Assay, Incubation, Flow Cytometry, Cytometry, Expressing, Clone Assay, Plasmid Preparation, Transformation Assay

    C/EBP-β LIP induces calreticulin expression and immunogenic cell death. MDA-MB-231 cells were left untreated (−, ctrl) or transfected with an empty pcDNA4/TO vector (em/empty), with a pcDNA4/TO expression vector encoding C/EBP-β LAP or C/EBP-β LIP, respectively. a. Whole cell lysates were probed with an antibody recognizing both C/EBP-β LAP and LIP isoforms. The expression of β-tubulin was used as control of equal protein loading. The figure is representative of 1 out of 3 experiments. b. ChIP was performed to evaluate the binding of LAP or LIP to the CRT promoter (sites: 831–843; 1302–1313). no Ab: no anti-C/EBP-β antibody; bl: blank; DNA input: genomic DNA. The figure is representative of 1 out of 3 experiments. c. The relative expression of CRT gene was measured in triplicates by qRT-PCR. Data are presented as means±SD (n = 3). * p

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Increasing intratumor C/EBP-β LIP and nitric oxide levels overcome resistance to doxorubicin in triple negative breast cancer

    doi: 10.1186/s13046-018-0967-0

    Figure Lengend Snippet: C/EBP-β LIP induces calreticulin expression and immunogenic cell death. MDA-MB-231 cells were left untreated (−, ctrl) or transfected with an empty pcDNA4/TO vector (em/empty), with a pcDNA4/TO expression vector encoding C/EBP-β LAP or C/EBP-β LIP, respectively. a. Whole cell lysates were probed with an antibody recognizing both C/EBP-β LAP and LIP isoforms. The expression of β-tubulin was used as control of equal protein loading. The figure is representative of 1 out of 3 experiments. b. ChIP was performed to evaluate the binding of LAP or LIP to the CRT promoter (sites: 831–843; 1302–1313). no Ab: no anti-C/EBP-β antibody; bl: blank; DNA input: genomic DNA. The figure is representative of 1 out of 3 experiments. c. The relative expression of CRT gene was measured in triplicates by qRT-PCR. Data are presented as means±SD (n = 3). * p

    Article Snippet: The pcDNA4/TO expression vectors (Invitrogen Life Technologies, Milan, Italy) for LAP and LIP, produced as reported previously [ ], were co-transduced with pcDNA6/TR vector (Invitrogen Life Technologies) in parental cells.

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Binding Assay, Quantitative RT-PCR

    Reduction of cell migration by over-expression of PEA3 in breast cancer cells is rescued by exogenous FGF-10 treatment. MDA-MB-231 and MCF-7 cells (1×10 5 ) transfected either with empty vector (white and light grey bars) or pcDNA4/TO-PEA3 (black and dark grey bars), were plated onto Transwell ® filters in 24-well plates 36 h post-transfection. Cells were incubated overnight in medium without FBS supplemented with 0.1% BSA and with (grey bars) or without (black and white bars) recombinant FGF-10 (100 ng/ml), where the lower surface of the membrane had been coated with fibronectin (black bars). After 12 h, for both assay conditions, significantly fewer PEA3-transfected cells had migrated to the lower surface, when compared to controls (*p

    Journal: European Journal of Cell Biology

    Article Title: Negative regulation of fibroblast growth factor 10 (FGF-10) by polyoma enhancer activator 3 (PEA3)

    doi: 10.1016/j.ejcb.2009.01.004

    Figure Lengend Snippet: Reduction of cell migration by over-expression of PEA3 in breast cancer cells is rescued by exogenous FGF-10 treatment. MDA-MB-231 and MCF-7 cells (1×10 5 ) transfected either with empty vector (white and light grey bars) or pcDNA4/TO-PEA3 (black and dark grey bars), were plated onto Transwell ® filters in 24-well plates 36 h post-transfection. Cells were incubated overnight in medium without FBS supplemented with 0.1% BSA and with (grey bars) or without (black and white bars) recombinant FGF-10 (100 ng/ml), where the lower surface of the membrane had been coated with fibronectin (black bars). After 12 h, for both assay conditions, significantly fewer PEA3-transfected cells had migrated to the lower surface, when compared to controls (*p

    Article Snippet: The hPEA3 PCR product was cloned into the BamHI and XbaI sites of the pcDNA4/TO mammalian expression vector (Invitrogen).

    Techniques: Migration, Over Expression, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Incubation, Recombinant

    Over-expression of PEA3 in MDA-MB-231 cells decreases Fgf-10 expression. Full-length human PEA3 cDNA (NM_001079675.1) was cloned into the pcDNA4/TO expression vector and transfected into the metastatic breast cancer cell line MDA-MB-231, with empty vector transfection serving as a control. PEA3 transfection resulted in a dramatic increase in both PEA3 mRNA (A) and protein (B) expression levels after 48 h. Concomitant with this, real-time RT-PCR showed that Fgf-10 expression was reduced to approximately 73% of the level measured in control cells (A), and this was reflected in a similar decrease (70%) in protein levels (B). HPRT and tubulin levels were measured as loading/quality controls for mRNA and protein, respectively. Real-time RT-PCR data were analysed using the 2 −ΔΔCt method. All quantitation represents data from at least three independent experiments, and reflects either real-time RT-PCR data (n=5; each in triplicate) (A) or Western blot densitometry (n=3) (B). Statistical significance was determined with Student's t -test. Results were considered significant at P

    Journal: European Journal of Cell Biology

    Article Title: Negative regulation of fibroblast growth factor 10 (FGF-10) by polyoma enhancer activator 3 (PEA3)

    doi: 10.1016/j.ejcb.2009.01.004

    Figure Lengend Snippet: Over-expression of PEA3 in MDA-MB-231 cells decreases Fgf-10 expression. Full-length human PEA3 cDNA (NM_001079675.1) was cloned into the pcDNA4/TO expression vector and transfected into the metastatic breast cancer cell line MDA-MB-231, with empty vector transfection serving as a control. PEA3 transfection resulted in a dramatic increase in both PEA3 mRNA (A) and protein (B) expression levels after 48 h. Concomitant with this, real-time RT-PCR showed that Fgf-10 expression was reduced to approximately 73% of the level measured in control cells (A), and this was reflected in a similar decrease (70%) in protein levels (B). HPRT and tubulin levels were measured as loading/quality controls for mRNA and protein, respectively. Real-time RT-PCR data were analysed using the 2 −ΔΔCt method. All quantitation represents data from at least three independent experiments, and reflects either real-time RT-PCR data (n=5; each in triplicate) (A) or Western blot densitometry (n=3) (B). Statistical significance was determined with Student's t -test. Results were considered significant at P

    Article Snippet: The hPEA3 PCR product was cloned into the BamHI and XbaI sites of the pcDNA4/TO mammalian expression vector (Invitrogen).

    Techniques: Over Expression, Multiple Displacement Amplification, Expressing, Clone Assay, Plasmid Preparation, Transfection, Quantitative RT-PCR, Quantitation Assay, Western Blot

    DJH1 joint sequences from MSH2 +/+ and MSH2 −/− littermates. DJH PCR products from the reaction shown in Figure 1B were used as templates in a nested PCR using a JH1–JH2 intronic primer (J1CY) to amplify only joints that utilized the JH1 segment. PCR products were cloned into the PCR2.1 vector and sequenced. ( A ) Upper panel shows sequences from littermate WT-1 and lower panel shows sequences from littermate WT-2. ( B ) Upper panel shows sequences from littermate MSH2 −/− 1 and lower panel shows sequences from littermate MSH2 −/− 2. Sequences followed by an asterisk were scored as joining by homology joints. P-nucleotides are indicated in bold.

    Journal: Nucleic Acids Research

    Article Title: Lack of MSH2 involvement differentiates V(D)J recombination from other non-homologous end joining events

    doi: 10.1093/nar/gki983

    Figure Lengend Snippet: DJH1 joint sequences from MSH2 +/+ and MSH2 −/− littermates. DJH PCR products from the reaction shown in Figure 1B were used as templates in a nested PCR using a JH1–JH2 intronic primer (J1CY) to amplify only joints that utilized the JH1 segment. PCR products were cloned into the PCR2.1 vector and sequenced. ( A ) Upper panel shows sequences from littermate WT-1 and lower panel shows sequences from littermate WT-2. ( B ) Upper panel shows sequences from littermate MSH2 −/− 1 and lower panel shows sequences from littermate MSH2 −/− 2. Sequences followed by an asterisk were scored as joining by homology joints. P-nucleotides are indicated in bold.

    Article Snippet: Of the same PCR product 4 µl was used in ligation with the PCR2.1 TA cloning vector (Invitrogen), and plated on Kanamycin plates containing X-gal.

    Techniques: Polymerase Chain Reaction, Nested PCR, Clone Assay, Plasmid Preparation

    DJH joint sequences from MSH2 +/+ and MSH2 −/− littermates. DJH PCR products from the reaction shown in Figure 1B were cloned into the PCR2.1 vector and sequenced. ( A ) Upper panel shows sequences from littermate WT-1 (MSH2 +/+ ) and lower panel shows sequences from littermate WT-2 (MSH2 +/+ ). ( B ) Upper panel shows sequences from littermate MSH2 −/− 1 and lower panel shows sequences from littermate MSH2 −/− 2. P-nucleotides are indicated in bold.

    Journal: Nucleic Acids Research

    Article Title: Lack of MSH2 involvement differentiates V(D)J recombination from other non-homologous end joining events

    doi: 10.1093/nar/gki983

    Figure Lengend Snippet: DJH joint sequences from MSH2 +/+ and MSH2 −/− littermates. DJH PCR products from the reaction shown in Figure 1B were cloned into the PCR2.1 vector and sequenced. ( A ) Upper panel shows sequences from littermate WT-1 (MSH2 +/+ ) and lower panel shows sequences from littermate WT-2 (MSH2 +/+ ). ( B ) Upper panel shows sequences from littermate MSH2 −/− 1 and lower panel shows sequences from littermate MSH2 −/− 2. P-nucleotides are indicated in bold.

    Article Snippet: Of the same PCR product 4 µl was used in ligation with the PCR2.1 TA cloning vector (Invitrogen), and plated on Kanamycin plates containing X-gal.

    Techniques: Polymerase Chain Reaction, Clone Assay, Plasmid Preparation

    Analysis of the DFL16.1/JH1 joints from the A-MuLV cell line transfection assay. ( A ) Western blot analysis of MSH2 expression in the A-MuLV cell lines used in the transfection assay: 15–63 is MSH2 +/− and 8–58 is MSH2 −/− . Upper panel was probed for MSH2 and lower panel was probed for β-actin as a loading control. ( B ) DNA recovered after transfection of 15–63 and 8–58 cell lines was used as PCR template using the DFS/J1CY primer pair. Products were cloned into the PCR2.1 vector and sequenced using the T7 promotor. Sequences followed by an asterisk were scored as joining by homology joints. P-nucleotides are indicated in bold.

    Journal: Nucleic Acids Research

    Article Title: Lack of MSH2 involvement differentiates V(D)J recombination from other non-homologous end joining events

    doi: 10.1093/nar/gki983

    Figure Lengend Snippet: Analysis of the DFL16.1/JH1 joints from the A-MuLV cell line transfection assay. ( A ) Western blot analysis of MSH2 expression in the A-MuLV cell lines used in the transfection assay: 15–63 is MSH2 +/− and 8–58 is MSH2 −/− . Upper panel was probed for MSH2 and lower panel was probed for β-actin as a loading control. ( B ) DNA recovered after transfection of 15–63 and 8–58 cell lines was used as PCR template using the DFS/J1CY primer pair. Products were cloned into the PCR2.1 vector and sequenced using the T7 promotor. Sequences followed by an asterisk were scored as joining by homology joints. P-nucleotides are indicated in bold.

    Article Snippet: Of the same PCR product 4 µl was used in ligation with the PCR2.1 TA cloning vector (Invitrogen), and plated on Kanamycin plates containing X-gal.

    Techniques: Transfection, Western Blot, Expressing, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation

    Purified rMG309c induced proinflammatory cytokine elaboration. (A) The largest coding fragment of MG309 (designated rMG309c) free of UGA codons (stop signal [St] in E. coli ) was PCR amplified and cloned into pPROEX-HTb for direct expression of a six-His-tagged

    Journal:

    Article Title: Mycoplasma genitalium-Encoded MG309 Activates NF-?B via Toll-Like Receptors 2 and 6 To Elicit Proinflammatory Cytokine Secretion from Human Genital Epithelial Cells ▿

    doi: 10.1128/IAI.00845-08

    Figure Lengend Snippet: Purified rMG309c induced proinflammatory cytokine elaboration. (A) The largest coding fragment of MG309 (designated rMG309c) free of UGA codons (stop signal [St] in E. coli ) was PCR amplified and cloned into pPROEX-HTb for direct expression of a six-His-tagged

    Article Snippet: Each PCR product was cloned into the pPROEX-HTb expression plasmid (Invitrogen), which directs expression of the recombinant protein with a six-histidine tag, allowing purification using a nickel affinity column.

    Techniques: Purification, Polymerase Chain Reaction, Amplification, Clone Assay, Expressing

    The level of anti-SjGST in serum samples after vaccination and after Schistosoma japonicum challenge. Serum samples were collected from the tails of mice immunized with pcDNA4 (white), pcDNA/SjGST (light grey), pcDNA/SjGST + pIL-12 (dark grey), pcDNA/SjGST + rSjGST (black), or pcDNA/SjGST + pIL-12 + rSjGST (mosaic), 8 weeks after vaccination (left panel) or 8 weeks after infection (right panel). The antibody titers of (A) total IgG, (B) IgG1, (C) IgG2a, and (D) IgG2b were measured by ELISA. The OD405 value of each group was directly shown in the figure. The results are shown as the mean ± standard deviation from three independent experiments. *: p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum

    doi: 10.1371/journal.pntd.0004459

    Figure Lengend Snippet: The level of anti-SjGST in serum samples after vaccination and after Schistosoma japonicum challenge. Serum samples were collected from the tails of mice immunized with pcDNA4 (white), pcDNA/SjGST (light grey), pcDNA/SjGST + pIL-12 (dark grey), pcDNA/SjGST + rSjGST (black), or pcDNA/SjGST + pIL-12 + rSjGST (mosaic), 8 weeks after vaccination (left panel) or 8 weeks after infection (right panel). The antibody titers of (A) total IgG, (B) IgG1, (C) IgG2a, and (D) IgG2b were measured by ELISA. The OD405 value of each group was directly shown in the figure. The results are shown as the mean ± standard deviation from three independent experiments. *: p

    Article Snippet: The gene products was digested with Bam HI/Eco RV at 37°C for 4 h, and then subcloned into the pcDNA4 vector (Invitrogen, Waltham, MA, USA) at 37°C overnight to generate the pcDNA/SjGST construct.

    Techniques: Mouse Assay, Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation

    The cytokine mRNA expression levels in spleen cells after infection. Spleen immune cells were isolated from the spleen of mice 8 weeks after infection. The mRNA expression levels of (A) IL-2, (B) IL-12, (C) IFN-γ, (D) IL-4, (E) IL-10, and (F) TNF-α were measured by qRT-PCR. Comparative CT was used to determine the individual relative mRNA expression levels (fold differences) when normalized to the expression level of the internal control genes, GAPDH and 18s. The cytokine gene expression level of the pcDNA4 group was used as background to determine the relative expression. The results are shown as the mean ± standard deviation from three independent experiments. *: p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum

    doi: 10.1371/journal.pntd.0004459

    Figure Lengend Snippet: The cytokine mRNA expression levels in spleen cells after infection. Spleen immune cells were isolated from the spleen of mice 8 weeks after infection. The mRNA expression levels of (A) IL-2, (B) IL-12, (C) IFN-γ, (D) IL-4, (E) IL-10, and (F) TNF-α were measured by qRT-PCR. Comparative CT was used to determine the individual relative mRNA expression levels (fold differences) when normalized to the expression level of the internal control genes, GAPDH and 18s. The cytokine gene expression level of the pcDNA4 group was used as background to determine the relative expression. The results are shown as the mean ± standard deviation from three independent experiments. *: p

    Article Snippet: The gene products was digested with Bam HI/Eco RV at 37°C for 4 h, and then subcloned into the pcDNA4 vector (Invitrogen, Waltham, MA, USA) at 37°C overnight to generate the pcDNA/SjGST construct.

    Techniques: Expressing, Infection, Isolation, Mouse Assay, Quantitative RT-PCR, Standard Deviation

    The worm and hepatic egg burdens of vaccinated animals challenged with Schistosoma japonicum . Six groups of mice were immunized with pcDNA4 vehicle, pcDNA/SjGST vaccine, pIL-12 and rSjGST. The vaccinated mice were infected with 30 cercaria/ mouse. Each group included 19–21 animals (with the exception of Group I, n = 8) from three independent experiments. Eight weeks after schistosome infection, (A) adult worms were collected from infected mice and counted under an anatomy microscope. Each point represents the number of worms in a single mouse. The horizontal black lane represents the mean ± SD of that group. NS: not significant; ***: p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Combined IL-12 Plasmid and Recombinant SjGST Enhance the Protective and Anti-pathology Effect of SjGST DNA Vaccine Against Schistosoma japonicum

    doi: 10.1371/journal.pntd.0004459

    Figure Lengend Snippet: The worm and hepatic egg burdens of vaccinated animals challenged with Schistosoma japonicum . Six groups of mice were immunized with pcDNA4 vehicle, pcDNA/SjGST vaccine, pIL-12 and rSjGST. The vaccinated mice were infected with 30 cercaria/ mouse. Each group included 19–21 animals (with the exception of Group I, n = 8) from three independent experiments. Eight weeks after schistosome infection, (A) adult worms were collected from infected mice and counted under an anatomy microscope. Each point represents the number of worms in a single mouse. The horizontal black lane represents the mean ± SD of that group. NS: not significant; ***: p

    Article Snippet: The gene products was digested with Bam HI/Eco RV at 37°C for 4 h, and then subcloned into the pcDNA4 vector (Invitrogen, Waltham, MA, USA) at 37°C overnight to generate the pcDNA/SjGST construct.

    Techniques: Mouse Assay, Infection, Microscopy

    miR-199a-3p suppressed RA-FLS proliferation ( A ) RT-qPCR of miR-199a-3p in RA-FLSs transfected with pSilencer 4.1-miR-199a-3p plasmid (miR-199a-3p) or no-insert control pSilencer 4.1-CMV puro plasmid (Mock). ( B ) Forty-eight hours post transfection, MTT assay was performed to test the viability of miR-199a-3p or Mock RA-FLSs. ( C , D ). EdU assay of miR-199a-3p or Mock transfected RA-FLSs 48 h post transfection. Cells were stained for EdU and Hoechst (to mark nuclei) (C), and quantitated for EdU+ cell percentage (D). CCK8 assay showed that ectopic expression of miR-199a-3p significantly inhibited RA-FLS proliferation rate over 5 days. n =4 or n =6 (A,D); * P

    Journal: Bioscience Reports

    Article Title: MiR-199a-3p inhibits proliferation and induces apoptosis in rheumatoid arthritis fibroblast-like synoviocytes via suppressing retinoblastoma 1

    doi: 10.1042/BSR20180982

    Figure Lengend Snippet: miR-199a-3p suppressed RA-FLS proliferation ( A ) RT-qPCR of miR-199a-3p in RA-FLSs transfected with pSilencer 4.1-miR-199a-3p plasmid (miR-199a-3p) or no-insert control pSilencer 4.1-CMV puro plasmid (Mock). ( B ) Forty-eight hours post transfection, MTT assay was performed to test the viability of miR-199a-3p or Mock RA-FLSs. ( C , D ). EdU assay of miR-199a-3p or Mock transfected RA-FLSs 48 h post transfection. Cells were stained for EdU and Hoechst (to mark nuclei) (C), and quantitated for EdU+ cell percentage (D). CCK8 assay showed that ectopic expression of miR-199a-3p significantly inhibited RA-FLS proliferation rate over 5 days. n =4 or n =6 (A,D); * P

    Article Snippet: DNA constructs and siRNA To generate the miR-199a-3p overexpressing construct, a DNA fragment containing human miR-199a-3p precursor was amplified by PCR from human genomic DNA and inserted into a pSilencer 4.1-CMV puro mammalian expression vector (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, MTT Assay, EdU Assay, Staining, CCK-8 Assay, Expressing

    RB1 is a direct target of miR-199a-3p ( A ) TargetScan analysis showing the WT 3′-UTR of RB1 mRNA containing a putative miR-199a-3p target site. A mutant (MUT) sequence was designed accordingly to be tested for luciferase assay together with the WT. ( B ) Luciferase reporter assay comparing WT with MUT RB1 3′-UTR targetting by miR-199a-3p. RA-FLSs cells were co-transfected with RB1 3′-UTR firefly luciferase reporter constructs harboring WT or MUT miR-199a-3p-targetting sequences and an miR-199a-3p-expressing plasmid (miR-199a-3p) or a pSliencer 4.1-CMV puro vector (miR-control). Firefly luciferase activity was normalized to Renilla luciferase activity. ( C ) RT-qPCR analysis of RB1 mRNA in RA-FLSs transfected with miR-199a-3p or miR-control. ( D ) RB1 protein expression in RA-FLSs transfected with miR-199a-3p or miR-control. n =5; * P

    Journal: Bioscience Reports

    Article Title: MiR-199a-3p inhibits proliferation and induces apoptosis in rheumatoid arthritis fibroblast-like synoviocytes via suppressing retinoblastoma 1

    doi: 10.1042/BSR20180982

    Figure Lengend Snippet: RB1 is a direct target of miR-199a-3p ( A ) TargetScan analysis showing the WT 3′-UTR of RB1 mRNA containing a putative miR-199a-3p target site. A mutant (MUT) sequence was designed accordingly to be tested for luciferase assay together with the WT. ( B ) Luciferase reporter assay comparing WT with MUT RB1 3′-UTR targetting by miR-199a-3p. RA-FLSs cells were co-transfected with RB1 3′-UTR firefly luciferase reporter constructs harboring WT or MUT miR-199a-3p-targetting sequences and an miR-199a-3p-expressing plasmid (miR-199a-3p) or a pSliencer 4.1-CMV puro vector (miR-control). Firefly luciferase activity was normalized to Renilla luciferase activity. ( C ) RT-qPCR analysis of RB1 mRNA in RA-FLSs transfected with miR-199a-3p or miR-control. ( D ) RB1 protein expression in RA-FLSs transfected with miR-199a-3p or miR-control. n =5; * P

    Article Snippet: DNA constructs and siRNA To generate the miR-199a-3p overexpressing construct, a DNA fragment containing human miR-199a-3p precursor was amplified by PCR from human genomic DNA and inserted into a pSilencer 4.1-CMV puro mammalian expression vector (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Techniques: Mutagenesis, Sequencing, Luciferase, Reporter Assay, Transfection, Construct, Expressing, Plasmid Preparation, Activity Assay, Quantitative RT-PCR

    miR-199a-3p regulated RA-FLS proliferation and apoptosis partially via suppressing RB1 RA-FLS was transfected with an miR-199a-3p-expressing plasmid (miR-199a-3p) or a pSliencer 4.1-CMV puro vector (miR-control) or miR-199a-3p in combination with an RB1-overexpression plasmid (miR + pcDNA-RB1) or miR-199a-3p in combination with a negative control plasmid (miR + pcDNA-NC). ( A ) Western blot of RB1 protein in RA-FLSs transfected with negative control (NC) or pcDNA3-RB1. ( B ) Proliferation of transfected RA-FLS was measured by the MTT assay. ( C ) Apoptosis was measured by FITC Annexin V and PI staining followed by flow cytometry. ( D ) Caspase-3 activity was measured by a colorimetric method. n =6; * P

    Journal: Bioscience Reports

    Article Title: MiR-199a-3p inhibits proliferation and induces apoptosis in rheumatoid arthritis fibroblast-like synoviocytes via suppressing retinoblastoma 1

    doi: 10.1042/BSR20180982

    Figure Lengend Snippet: miR-199a-3p regulated RA-FLS proliferation and apoptosis partially via suppressing RB1 RA-FLS was transfected with an miR-199a-3p-expressing plasmid (miR-199a-3p) or a pSliencer 4.1-CMV puro vector (miR-control) or miR-199a-3p in combination with an RB1-overexpression plasmid (miR + pcDNA-RB1) or miR-199a-3p in combination with a negative control plasmid (miR + pcDNA-NC). ( A ) Western blot of RB1 protein in RA-FLSs transfected with negative control (NC) or pcDNA3-RB1. ( B ) Proliferation of transfected RA-FLS was measured by the MTT assay. ( C ) Apoptosis was measured by FITC Annexin V and PI staining followed by flow cytometry. ( D ) Caspase-3 activity was measured by a colorimetric method. n =6; * P

    Article Snippet: DNA constructs and siRNA To generate the miR-199a-3p overexpressing construct, a DNA fragment containing human miR-199a-3p precursor was amplified by PCR from human genomic DNA and inserted into a pSilencer 4.1-CMV puro mammalian expression vector (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Techniques: Transfection, Expressing, Plasmid Preparation, Over Expression, Negative Control, Western Blot, MTT Assay, Staining, Flow Cytometry, Cytometry, Activity Assay

    miR390 Expression Responds to Auxin during Lateral Root Induction. (A) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type plants during a time course of 10 μM auxin (IAA) treatment after 24 h of 10 μM NPA pretreatment. The blot was successively probed with DNA complementary to miR390, miR156, and miR160. U6 snRNA served as a loading control. (B) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type plants. Plants were pretreated with 10 μM NPA and then for another 24 h with either DMSO (–), 10 μM cycloheximide (CHX), 10 μM IAA, or both (CHX+IAA). U6 snRNA served as a loading control. (C) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type or mir390a plants treated (+) or untreated (−) for 24 h with 10 μM IAA after 24 h of 10 μM NPA pretreatment. In (A) to (C) , numbers are the ratios of miR390 to U6 signal. These experiments were done twice with similar results. (D) RT-PCR analysis of root RNA from 10-d-old wild-type plants treated for 24 h with 10 μM IAA (+) or untreated (−) after NPA pretreatment. Primers amplify specifically the MIR390a precursor and ACTIN2 (loading control) from cDNA (RT+) but not from genomic DNA (RT−). (E) and (F) Confocal analysis of pMIR390a:GUS-GFP expression in 10-d-old wild-type plants treated (+IAA) or untreated (Control) for 10 h with 10 μM IAA after NPA pretreatment. GFP is in green, and cell walls are stained by propidium iodide in red. Bars = 50 μm.

    Journal: The Plant Cell

    Article Title: miR390, Arabidopsis TAS3 tasiRNAs, and Their AUXIN RESPONSE FACTOR Targets Define an Autoregulatory Network Quantitatively Regulating Lateral Root Growth [W]

    doi: 10.1105/tpc.109.072553

    Figure Lengend Snippet: miR390 Expression Responds to Auxin during Lateral Root Induction. (A) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type plants during a time course of 10 μM auxin (IAA) treatment after 24 h of 10 μM NPA pretreatment. The blot was successively probed with DNA complementary to miR390, miR156, and miR160. U6 snRNA served as a loading control. (B) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type plants. Plants were pretreated with 10 μM NPA and then for another 24 h with either DMSO (–), 10 μM cycloheximide (CHX), 10 μM IAA, or both (CHX+IAA). U6 snRNA served as a loading control. (C) RNA gel blot analysis of 15 μg of root RNA from 10-d-old wild-type or mir390a plants treated (+) or untreated (−) for 24 h with 10 μM IAA after 24 h of 10 μM NPA pretreatment. In (A) to (C) , numbers are the ratios of miR390 to U6 signal. These experiments were done twice with similar results. (D) RT-PCR analysis of root RNA from 10-d-old wild-type plants treated for 24 h with 10 μM IAA (+) or untreated (−) after NPA pretreatment. Primers amplify specifically the MIR390a precursor and ACTIN2 (loading control) from cDNA (RT+) but not from genomic DNA (RT−). (E) and (F) Confocal analysis of pMIR390a:GUS-GFP expression in 10-d-old wild-type plants treated (+IAA) or untreated (Control) for 10 h with 10 μM IAA after NPA pretreatment. GFP is in green, and cell walls are stained by propidium iodide in red. Bars = 50 μm.

    Article Snippet: For quantitative RT-PCR analysis, 4 μg of total RNA was treated with RNase-free DNase (Fermentas) and reverse transcribed (Superscript II; Invitrogen). cDNA was diluted four times and used for amplification.

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

    EBV miR-BHRF1-2-5p, miR-BART2-5p, and cellular miR-17 regulate the latent to lytic switch. A and C-G. MutuI cells were transduced with pLCE control vector or sponge inhibitors to indicated EBV or cellular miRNAs, then treated for 24 or 42 hrs with 5 ug/mL anti-IgM. Total RNA was harvested and assayed by qRT-PCR for EBV gene expression as indicated. Reported are the averages of two independent experiments with qPCR performed in duplicate; expression levels are normalized to GAPDH and shown relative to mock treated cells (harvested at 42 hrs) for each miRNA sponge inhibitor. B. miRNA levels in anti-IgM treated, sponged MutuI cells assayed by qRT-PCR. Values are normalized to miR-16 and shown relative to pLCE control cells for each respective sponge inhibitor. Reported are the averages of two independent experiments with qPCR performed in duplicate. *By Student’s t test, p

    Journal: PLoS Pathogens

    Article Title: Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation

    doi: 10.1371/journal.ppat.1007535

    Figure Lengend Snippet: EBV miR-BHRF1-2-5p, miR-BART2-5p, and cellular miR-17 regulate the latent to lytic switch. A and C-G. MutuI cells were transduced with pLCE control vector or sponge inhibitors to indicated EBV or cellular miRNAs, then treated for 24 or 42 hrs with 5 ug/mL anti-IgM. Total RNA was harvested and assayed by qRT-PCR for EBV gene expression as indicated. Reported are the averages of two independent experiments with qPCR performed in duplicate; expression levels are normalized to GAPDH and shown relative to mock treated cells (harvested at 42 hrs) for each miRNA sponge inhibitor. B. miRNA levels in anti-IgM treated, sponged MutuI cells assayed by qRT-PCR. Values are normalized to miR-16 and shown relative to pLCE control cells for each respective sponge inhibitor. Reported are the averages of two independent experiments with qPCR performed in duplicate. *By Student’s t test, p

    Article Snippet: HEK293T cells plated in 96-well black-well plates were co-transfected with 20 ng of 3’UTR reporter and 250 ng of control vector (pLCE) or EBV miRNA expression vector [ , , ] using Lipofectamine2000 (Thermofisher) according to the manufacturer’s instructions.

    Techniques: Transduction, Plasmid Preparation, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction

    Validation of EBV miRNA targets. A-C. 293T cells were co-transfected with indicated 3’UTR luciferase reporters or psiCheck2 empty vector (C.) and EBV miRNA expression vectors (pLCE-based). 48–72 hrs post-transfection, cells were lysed and assayed for dual luciferase activity. PAR-CLIP interactions between EBV miRNAs and 3’UTRs are highlighted. SM = seed match. Reported are the averages of at least three independent experiments performed in triplicate. *By Student’s t-test, p

    Journal: PLoS Pathogens

    Article Title: Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation

    doi: 10.1371/journal.ppat.1007535

    Figure Lengend Snippet: Validation of EBV miRNA targets. A-C. 293T cells were co-transfected with indicated 3’UTR luciferase reporters or psiCheck2 empty vector (C.) and EBV miRNA expression vectors (pLCE-based). 48–72 hrs post-transfection, cells were lysed and assayed for dual luciferase activity. PAR-CLIP interactions between EBV miRNAs and 3’UTRs are highlighted. SM = seed match. Reported are the averages of at least three independent experiments performed in triplicate. *By Student’s t-test, p

    Article Snippet: HEK293T cells plated in 96-well black-well plates were co-transfected with 20 ng of 3’UTR reporter and 250 ng of control vector (pLCE) or EBV miRNA expression vector [ , , ] using Lipofectamine2000 (Thermofisher) according to the manufacturer’s instructions.

    Techniques: Transfection, Luciferase, Plasmid Preparation, Expressing, Activity Assay, Cross-linking Immunoprecipitation

    shRNAs to RAC1, GRB2, or SOS1 phenocopy EBV BHRF1-2 miRNA activity. A. BJAB cells were transduced with pL-mCherry or shRNAs to GRB2, PLCG1, or SOS1 as indicated. Lysates were analyzed by Western blot. Gapdh levels are shown as loading controls. Lysate from BJAB cells transduced with pLCE-BHRF1-2 was included in the Grb2 Western blot. B. Knockdown of individual target genes in BJAB-NFkB-GL4.32 cells was assayed by qRT-PCR analysis. Expression levels are normalized to GAPDH and reported relative to control (mCherry) cells. qPCR was performed in duplicate. C. Individual shRNAs were stably expressed in BJAB-NFkB-GL4.32 cells. Cells were stimulated for 18 hr with 5 ug/ml anti-IgM, then lysed and assayed for NF-kappaB responsive luciferase activity. NF-kappaB activity levels are normalized to mock treated cells. Averages and standard deviations (S.D.) are from two independent experiments performed in quadruplicate. By Student’s t-test, *p

    Journal: PLoS Pathogens

    Article Title: Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation

    doi: 10.1371/journal.ppat.1007535

    Figure Lengend Snippet: shRNAs to RAC1, GRB2, or SOS1 phenocopy EBV BHRF1-2 miRNA activity. A. BJAB cells were transduced with pL-mCherry or shRNAs to GRB2, PLCG1, or SOS1 as indicated. Lysates were analyzed by Western blot. Gapdh levels are shown as loading controls. Lysate from BJAB cells transduced with pLCE-BHRF1-2 was included in the Grb2 Western blot. B. Knockdown of individual target genes in BJAB-NFkB-GL4.32 cells was assayed by qRT-PCR analysis. Expression levels are normalized to GAPDH and reported relative to control (mCherry) cells. qPCR was performed in duplicate. C. Individual shRNAs were stably expressed in BJAB-NFkB-GL4.32 cells. Cells were stimulated for 18 hr with 5 ug/ml anti-IgM, then lysed and assayed for NF-kappaB responsive luciferase activity. NF-kappaB activity levels are normalized to mock treated cells. Averages and standard deviations (S.D.) are from two independent experiments performed in quadruplicate. By Student’s t-test, *p

    Article Snippet: HEK293T cells plated in 96-well black-well plates were co-transfected with 20 ng of 3’UTR reporter and 250 ng of control vector (pLCE) or EBV miRNA expression vector [ , , ] using Lipofectamine2000 (Thermofisher) according to the manufacturer’s instructions.

    Techniques: Activity Assay, Transduction, Western Blot, Quantitative RT-PCR, Expressing, Real-time Polymerase Chain Reaction, Stable Transfection, Luciferase

    EBV miR-BHRF1-2-5p contributes to the growth of EBV+ DLBCL cells. A. Taqman qRT-PCR analysis of miR-BHRF1-2-5p (5p) and miR-BHRF1-2-3p (3p) expression in miR-BHRF1-2-5p sponged DLBCLs (IBL1 and BCKN1). Values are normalized to U6 and reported relative to the BHRF1-2 miRNA levels in each respective DLBCL transduced with pLCE-CXCR4 control vector. B. Proliferation of DLBCLs as determined by MTT assay following stable transduction with pLCE-CXCR4 control vector or the miR-BHRF1-2-5p sponge. FAC-sorted, GFP+ DLBCLs were maintained in media containing 15% FBS and split one day prior to MTT assays. Values at Tn are normalized to the absorbance values at 0 hr (A-T0). Error bars represent S.D. of measurements from six or eight wells. *By Student’s t test, p

    Journal: PLoS Pathogens

    Article Title: Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation

    doi: 10.1371/journal.ppat.1007535

    Figure Lengend Snippet: EBV miR-BHRF1-2-5p contributes to the growth of EBV+ DLBCL cells. A. Taqman qRT-PCR analysis of miR-BHRF1-2-5p (5p) and miR-BHRF1-2-3p (3p) expression in miR-BHRF1-2-5p sponged DLBCLs (IBL1 and BCKN1). Values are normalized to U6 and reported relative to the BHRF1-2 miRNA levels in each respective DLBCL transduced with pLCE-CXCR4 control vector. B. Proliferation of DLBCLs as determined by MTT assay following stable transduction with pLCE-CXCR4 control vector or the miR-BHRF1-2-5p sponge. FAC-sorted, GFP+ DLBCLs were maintained in media containing 15% FBS and split one day prior to MTT assays. Values at Tn are normalized to the absorbance values at 0 hr (A-T0). Error bars represent S.D. of measurements from six or eight wells. *By Student’s t test, p

    Article Snippet: HEK293T cells plated in 96-well black-well plates were co-transfected with 20 ng of 3’UTR reporter and 250 ng of control vector (pLCE) or EBV miRNA expression vector [ , , ] using Lipofectamine2000 (Thermofisher) according to the manufacturer’s instructions.

    Techniques: Quantitative RT-PCR, Expressing, Transduction, Plasmid Preparation, MTT Assay

    Cellular targets of EBV miRNAs involved in BCR signaling pathways. ). Red boxes indicate presence of an interaction site for a specific 3’UTR. B.-D. Pathways downstream of the BCR are targeted by EBV miRNAs. Cellular targets are highlighted in pink with the corresponding EBV miRNA(s) listed in red. Pathways were constructed in PathVisio 3.0.

    Journal: PLoS Pathogens

    Article Title: Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation

    doi: 10.1371/journal.ppat.1007535

    Figure Lengend Snippet: Cellular targets of EBV miRNAs involved in BCR signaling pathways. ). Red boxes indicate presence of an interaction site for a specific 3’UTR. B.-D. Pathways downstream of the BCR are targeted by EBV miRNAs. Cellular targets are highlighted in pink with the corresponding EBV miRNA(s) listed in red. Pathways were constructed in PathVisio 3.0.

    Article Snippet: HEK293T cells plated in 96-well black-well plates were co-transfected with 20 ng of 3’UTR reporter and 250 ng of control vector (pLCE) or EBV miRNA expression vector [ , , ] using Lipofectamine2000 (Thermofisher) according to the manufacturer’s instructions.

    Techniques: Construct

    EBV miRNAs disrupt BCR-mediated signaling events. A. BJAB-NFkB-GL4.32, stably transfected with a NF-kappaB-responsive firefly luciferase reporter, were transduced with pLCE empty vector or individual EBV miRNA vectors. Cells were plated in 96-well black-well plates, treated with 10 ug/mL anti-IgM for 18 hrs, and analyzed for luciferase activity. Values are normalized to mock treated cells and reported relative to pLCE control. Shown are the averages of three independent experiments performed in quadruplicate. B. BJAB-NFkBLuc cells, expressing a NF-kappaB-responsive firefly luciferase reporter and a renilla luciferase reporter for internal control, were transduced with pLCE empty vector or individual EBV miRNA vectors. Cells were treated with 5 ug/mL anti-IgM for 18 hr, and then lysed in 1X passive lysis buffer. Luciferase activity was measured using the dual luciferase reporter kit. Values are reported relative to mock-treated control (pLCE) cells. Shown are the averages of four independent experiments performed in triplicate. C. BJAB-NFkBLuc cells transduced with pLCE or pLCE-BHRF1-2 were stimulated with 100 ng/mL LPS for 6 hrs, then analyzed for luciferase activity as in (A.). Shown are the averages of three independent experiments performed in triplicate. D. and E. BJAB-AP1-GL4.44 cells, expressing an AP1-responsive firefly luciferase reporter, were transduced with pLCE-based EBV miRNA expression vectors, treated with 10 ug/mL anti-IgM for 6 hrs (D.) or 18 hrs (E.), then analyzed for luciferase activity. Values are normalized to mock treated cells. Shown are the averages of three independent experiments performed in quadruplicate. F. EBV miRNA expression. RNA was harvested from BJAB cells transduced with control vector (pLCE) or individual EBV miRNA expression vectors (pLCE-miR) and Mutu I cells treated with 5 ug/mL anti-IgM for 48 hrs. miRNAs were detected by qRT-PCR. Values are normalized to cellular miR-16 and reported relative to levels in Mutu I cells. By Student’s t-test, *p

    Journal: PLoS Pathogens

    Article Title: Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation

    doi: 10.1371/journal.ppat.1007535

    Figure Lengend Snippet: EBV miRNAs disrupt BCR-mediated signaling events. A. BJAB-NFkB-GL4.32, stably transfected with a NF-kappaB-responsive firefly luciferase reporter, were transduced with pLCE empty vector or individual EBV miRNA vectors. Cells were plated in 96-well black-well plates, treated with 10 ug/mL anti-IgM for 18 hrs, and analyzed for luciferase activity. Values are normalized to mock treated cells and reported relative to pLCE control. Shown are the averages of three independent experiments performed in quadruplicate. B. BJAB-NFkBLuc cells, expressing a NF-kappaB-responsive firefly luciferase reporter and a renilla luciferase reporter for internal control, were transduced with pLCE empty vector or individual EBV miRNA vectors. Cells were treated with 5 ug/mL anti-IgM for 18 hr, and then lysed in 1X passive lysis buffer. Luciferase activity was measured using the dual luciferase reporter kit. Values are reported relative to mock-treated control (pLCE) cells. Shown are the averages of four independent experiments performed in triplicate. C. BJAB-NFkBLuc cells transduced with pLCE or pLCE-BHRF1-2 were stimulated with 100 ng/mL LPS for 6 hrs, then analyzed for luciferase activity as in (A.). Shown are the averages of three independent experiments performed in triplicate. D. and E. BJAB-AP1-GL4.44 cells, expressing an AP1-responsive firefly luciferase reporter, were transduced with pLCE-based EBV miRNA expression vectors, treated with 10 ug/mL anti-IgM for 6 hrs (D.) or 18 hrs (E.), then analyzed for luciferase activity. Values are normalized to mock treated cells. Shown are the averages of three independent experiments performed in quadruplicate. F. EBV miRNA expression. RNA was harvested from BJAB cells transduced with control vector (pLCE) or individual EBV miRNA expression vectors (pLCE-miR) and Mutu I cells treated with 5 ug/mL anti-IgM for 48 hrs. miRNAs were detected by qRT-PCR. Values are normalized to cellular miR-16 and reported relative to levels in Mutu I cells. By Student’s t-test, *p

    Article Snippet: HEK293T cells plated in 96-well black-well plates were co-transfected with 20 ng of 3’UTR reporter and 250 ng of control vector (pLCE) or EBV miRNA expression vector [ , , ] using Lipofectamine2000 (Thermofisher) according to the manufacturer’s instructions.

    Techniques: Stable Transfection, Transfection, Luciferase, Transduction, Plasmid Preparation, Activity Assay, Expressing, Lysis, Quantitative RT-PCR

    Hypothetical model by which EBV miRNAs, such as miR-BHRF1-2-5p, regulate signal transduction components downstream of BCR and modulate the latent/lytic switch. A. EBV miR-BHRF1-2-5p regulates Grb2 protein levels in multiple EBV-infected B cell types which contributes to B cell proliferation (irrespective of an intact BCR). B. Multiple EBV miRNAs, including miR-BHRF1-2-5p, attenuate signaling through the BCR. Disruption of EBV miR-BHRF1-2-5p and miR-BART2-5p activities, in particular, impact the amplitude of virus reactivation when triggered by antigen cross-linking.

    Journal: PLoS Pathogens

    Article Title: Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation

    doi: 10.1371/journal.ppat.1007535

    Figure Lengend Snippet: Hypothetical model by which EBV miRNAs, such as miR-BHRF1-2-5p, regulate signal transduction components downstream of BCR and modulate the latent/lytic switch. A. EBV miR-BHRF1-2-5p regulates Grb2 protein levels in multiple EBV-infected B cell types which contributes to B cell proliferation (irrespective of an intact BCR). B. Multiple EBV miRNAs, including miR-BHRF1-2-5p, attenuate signaling through the BCR. Disruption of EBV miR-BHRF1-2-5p and miR-BART2-5p activities, in particular, impact the amplitude of virus reactivation when triggered by antigen cross-linking.

    Article Snippet: HEK293T cells plated in 96-well black-well plates were co-transfected with 20 ng of 3’UTR reporter and 250 ng of control vector (pLCE) or EBV miRNA expression vector [ , , ] using Lipofectamine2000 (Thermofisher) according to the manufacturer’s instructions.

    Techniques: Transduction, Infection

    PDCD4 as negative regulator of dermal fibroblast senescence and CAF activation A. Clonogenecity assays. HDFs were infected with two shRNA PDCD4-silencing lentiviruses in parallel with empty vector control, followed by selection with puromycin for 48hrs, then plated under low density conditions on triplicated dishes (1000 cells per 60mm dish). Colony formation was measured by crystal violet staining after 14 days. B. Senescence Associated β-galactosidase activity (SA β-gal) assay. HDFs infected with the PDCD4-silencing and control lentiviruses as in the previous panel were stained with SA β-gal at 7 days after infection. Representative images (left panel) and quantification of positive SA β-gal staining in ∼100 cells (right panel) are shown. C. Parallel cultures of cells as in the previous panel were analyzed for expression of PDCD4 gene silencing and expression of the senescence-determinant CDKN1A gene by RT-PCR. D. , E. HDFs were transfected with two different PDCD4-silencing siRNAs in parallel with scrambled siRNA control, followed, 72hrs after transfection, by analysis of expression of the indicated genes by real time RT-PCR. Insert: Immunoblot analysis of HDFs plus/minus siRNA-mediated silencing of PDCD4. F. HDFs, Saos-2 cells, human primary keratinocytes (HKCs) and HeLa cells were transfected with siRNAs for silencing of the CSL or PDCD4 genes in parallel with scrambled siRNA controls, followed by determination of HES1 expression by real time RT-PCR. G. , H. Immunoblot analysis of CSL and PDCD4 expression in several HDF and HKC strains and Saos-2 and HeLa cells as indicated, with tubulin as equal loading control.

    Journal: Oncotarget

    Article Title: PDCD4 is a CSL associated protein with a transcription repressive function in cancer associated fibroblast activation

    doi: 10.18632/oncotarget.11227

    Figure Lengend Snippet: PDCD4 as negative regulator of dermal fibroblast senescence and CAF activation A. Clonogenecity assays. HDFs were infected with two shRNA PDCD4-silencing lentiviruses in parallel with empty vector control, followed by selection with puromycin for 48hrs, then plated under low density conditions on triplicated dishes (1000 cells per 60mm dish). Colony formation was measured by crystal violet staining after 14 days. B. Senescence Associated β-galactosidase activity (SA β-gal) assay. HDFs infected with the PDCD4-silencing and control lentiviruses as in the previous panel were stained with SA β-gal at 7 days after infection. Representative images (left panel) and quantification of positive SA β-gal staining in ∼100 cells (right panel) are shown. C. Parallel cultures of cells as in the previous panel were analyzed for expression of PDCD4 gene silencing and expression of the senescence-determinant CDKN1A gene by RT-PCR. D. , E. HDFs were transfected with two different PDCD4-silencing siRNAs in parallel with scrambled siRNA control, followed, 72hrs after transfection, by analysis of expression of the indicated genes by real time RT-PCR. Insert: Immunoblot analysis of HDFs plus/minus siRNA-mediated silencing of PDCD4. F. HDFs, Saos-2 cells, human primary keratinocytes (HKCs) and HeLa cells were transfected with siRNAs for silencing of the CSL or PDCD4 genes in parallel with scrambled siRNA controls, followed by determination of HES1 expression by real time RT-PCR. G. , H. Immunoblot analysis of CSL and PDCD4 expression in several HDF and HKC strains and Saos-2 and HeLa cells as indicated, with tubulin as equal loading control.

    Article Snippet: The lentiviral vectors encoding shRNA against PDCD4 (TRCN0000059081 and TRCN0000059078) were purchased from Openbiosystem.

    Techniques: Activation Assay, Infection, shRNA, Plasmid Preparation, Selection, Staining, Activity Assay, β-Gal Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Quantitative RT-PCR

    Convergent binding of PDCD4 and CSL to common target genes A. HDFs were assayed by ChIP with anti-CSL and PDCD4 antibodies in parallel with non-immune IgGs followed by real-time PCR of the indicated promoter regions of the PTGS2, IL6, and HES1 gene to which CSL was previously shown to bind [ 3 ]. Similar results were obtained in a second independent experiment ( Supplementary Figure 1 ). B. Similar ChIP assays were performed with Saos-2 cells followed by PCR amplification of the indicated regions of the HES1 promoter. C. HDFs plus/minus shRNA-mediated CSL gene silencing were analyzed by ChIP with anti-PDCD4 antibodies in parallel with non-immune IgGs, followed by PCR of the indicated promoter regions of the HES1 and IL6 genes. D. , E. HDFs plus/minus siRNA-mediated PDCD4 gene silencing were analyzed, 72hrs after siRNA transfection, by ChIP with anti-CSL (D) or anti-H3K27Ac (E) antibodies in parallel with non-immune IgGs followed by PCR of the indicated promoter regions of the HES1 and IL6 genes.

    Journal: Oncotarget

    Article Title: PDCD4 is a CSL associated protein with a transcription repressive function in cancer associated fibroblast activation

    doi: 10.18632/oncotarget.11227

    Figure Lengend Snippet: Convergent binding of PDCD4 and CSL to common target genes A. HDFs were assayed by ChIP with anti-CSL and PDCD4 antibodies in parallel with non-immune IgGs followed by real-time PCR of the indicated promoter regions of the PTGS2, IL6, and HES1 gene to which CSL was previously shown to bind [ 3 ]. Similar results were obtained in a second independent experiment ( Supplementary Figure 1 ). B. Similar ChIP assays were performed with Saos-2 cells followed by PCR amplification of the indicated regions of the HES1 promoter. C. HDFs plus/minus shRNA-mediated CSL gene silencing were analyzed by ChIP with anti-PDCD4 antibodies in parallel with non-immune IgGs, followed by PCR of the indicated promoter regions of the HES1 and IL6 genes. D. , E. HDFs plus/minus siRNA-mediated PDCD4 gene silencing were analyzed, 72hrs after siRNA transfection, by ChIP with anti-CSL (D) or anti-H3K27Ac (E) antibodies in parallel with non-immune IgGs followed by PCR of the indicated promoter regions of the HES1 and IL6 genes.

    Article Snippet: The lentiviral vectors encoding shRNA against PDCD4 (TRCN0000059081 and TRCN0000059078) were purchased from Openbiosystem.

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, shRNA, Transfection

    Dermal fibroblasts with PDCD4 gene silencing promote formation of SCC with higher proliferative index and suppressed differentiation A. Immunofluorescence of ear lesions formed by SCC13 cells with HDFs plus/minus shRNA-mediated silencing of PDCD4 with antibodies against Ki67 and Vimentin (upper panels) or Involucrin and p63 (lower panels). Shown are representative images as well as a quantification of Ki67, p63, and involucrin staining. Percentages of Ki67 positive nuclei in Vimentin negative SCC cells were quantified by Adobe Photoshop software using Lasso tool in three different fields from 3 ear pairs. p63 positive nuclei over total nuclei were quantified using Image J. *

    Journal: Oncotarget

    Article Title: PDCD4 is a CSL associated protein with a transcription repressive function in cancer associated fibroblast activation

    doi: 10.18632/oncotarget.11227

    Figure Lengend Snippet: Dermal fibroblasts with PDCD4 gene silencing promote formation of SCC with higher proliferative index and suppressed differentiation A. Immunofluorescence of ear lesions formed by SCC13 cells with HDFs plus/minus shRNA-mediated silencing of PDCD4 with antibodies against Ki67 and Vimentin (upper panels) or Involucrin and p63 (lower panels). Shown are representative images as well as a quantification of Ki67, p63, and involucrin staining. Percentages of Ki67 positive nuclei in Vimentin negative SCC cells were quantified by Adobe Photoshop software using Lasso tool in three different fields from 3 ear pairs. p63 positive nuclei over total nuclei were quantified using Image J. *

    Article Snippet: The lentiviral vectors encoding shRNA against PDCD4 (TRCN0000059081 and TRCN0000059078) were purchased from Openbiosystem.

    Techniques: Immunofluorescence, shRNA, Staining, Software

    Endogenous PDCD4/CSL association and co-localization A. Nuclear extracts from early passage HDFs were analyzed by immune-precipitation (IP) with anti-PDCD4 antibodies or non-immune IgGs followed by immunoblotting with antibodies against CSL or PDCD4 as indicated. Shown are the results of two different experiments with HDF strains of independent origin. B. Proximity ligation assay (PLA) were used for in situ detection of CSL-PDCD4 association in HDFs. Red fluorescence foci (PLA signals) represent the interaction between CSL and PDCD4, and were analyzed by confocal microscopy with DAPI staining of nuclei (blue). The specificity of CSL-PDCD4 PLA signals was confirmed by the significant reduction of PLA signal in HDFs with siRNA-mediated CSL gene silencing. Shown are representative PLA images and the average number of dots per nucleus in HDFs plus/minus siRNA-mediated CSL gene silencing. PLA dots were counted from at least 30 cells in four fields. * p

    Journal: Oncotarget

    Article Title: PDCD4 is a CSL associated protein with a transcription repressive function in cancer associated fibroblast activation

    doi: 10.18632/oncotarget.11227

    Figure Lengend Snippet: Endogenous PDCD4/CSL association and co-localization A. Nuclear extracts from early passage HDFs were analyzed by immune-precipitation (IP) with anti-PDCD4 antibodies or non-immune IgGs followed by immunoblotting with antibodies against CSL or PDCD4 as indicated. Shown are the results of two different experiments with HDF strains of independent origin. B. Proximity ligation assay (PLA) were used for in situ detection of CSL-PDCD4 association in HDFs. Red fluorescence foci (PLA signals) represent the interaction between CSL and PDCD4, and were analyzed by confocal microscopy with DAPI staining of nuclei (blue). The specificity of CSL-PDCD4 PLA signals was confirmed by the significant reduction of PLA signal in HDFs with siRNA-mediated CSL gene silencing. Shown are representative PLA images and the average number of dots per nucleus in HDFs plus/minus siRNA-mediated CSL gene silencing. PLA dots were counted from at least 30 cells in four fields. * p

    Article Snippet: The lentiviral vectors encoding shRNA against PDCD4 (TRCN0000059081 and TRCN0000059078) were purchased from Openbiosystem.

    Techniques: Proximity Ligation Assay, In Situ, Fluorescence, Confocal Microscopy, Staining