pcr4 expression vector  (Thermo Fisher)


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    Structured Review

    Thermo Fisher pcr4 expression vector
    Pcr4 Expression Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr4 expression vector/product/Thermo Fisher
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr4 expression vector - by Bioz Stars, 2020-05
    91/100 stars

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    Clone Assay:

    Article Title: Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants*
    Article Snippet: .. Purified PCR reactions products were cloned into the pCR4® expression vector (Life Technologies), using a TOPO® TA cloning kit (Life Technologies). .. Chemically-competent OneShot® TOP10 bacteria cells (Life Technologies) were used for superior transformation efficiency.

    TA Cloning:

    Article Title: Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants*
    Article Snippet: .. Purified PCR reactions products were cloned into the pCR4® expression vector (Life Technologies), using a TOPO® TA cloning kit (Life Technologies). .. Chemically-competent OneShot® TOP10 bacteria cells (Life Technologies) were used for superior transformation efficiency.

    Purification:

    Article Title: Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants*
    Article Snippet: .. Purified PCR reactions products were cloned into the pCR4® expression vector (Life Technologies), using a TOPO® TA cloning kit (Life Technologies). .. Chemically-competent OneShot® TOP10 bacteria cells (Life Technologies) were used for superior transformation efficiency.

    Expressing:

    Article Title: Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants*
    Article Snippet: .. Purified PCR reactions products were cloned into the pCR4® expression vector (Life Technologies), using a TOPO® TA cloning kit (Life Technologies). .. Chemically-competent OneShot® TOP10 bacteria cells (Life Technologies) were used for superior transformation efficiency.

    Polymerase Chain Reaction:

    Article Title: Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants*
    Article Snippet: .. Purified PCR reactions products were cloned into the pCR4® expression vector (Life Technologies), using a TOPO® TA cloning kit (Life Technologies). .. Chemically-competent OneShot® TOP10 bacteria cells (Life Technologies) were used for superior transformation efficiency.

    Plasmid Preparation:

    Article Title: Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants*
    Article Snippet: .. Purified PCR reactions products were cloned into the pCR4® expression vector (Life Technologies), using a TOPO® TA cloning kit (Life Technologies). .. Chemically-competent OneShot® TOP10 bacteria cells (Life Technologies) were used for superior transformation efficiency.

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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 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ebv mirna expression vector/product/Thermo Fisher
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    ebv mirna expression vector - by Bioz Stars, 2020-05
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    92
    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 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/psilencer 4 1 cmv puro mammalian expression vector/product/Thermo Fisher
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    psilencer 4 1 cmv puro mammalian expression vector - by Bioz Stars, 2020-05
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    91
    Thermo Fisher doxycycline inducible atf3 expression
    <t>Atf3</t> deletion in mouse dermal fibroblasts enhances tumorigenic behavior of keratinocyte-derived SCC. (A) RT-qPCR analysis of the indicated genes in multiple parallel cultures of primary dermal fibroblasts derived from either individual or pooled newborn Atf3 −/− ( n = 8) and Atf3 +/+ ( n = 5) mice. Values are expressed as log10 ratios in Atf3 −/− versus Atf3 +/+ cultures. Data are mean ± SEM, one-tailed one-sample t test, *, P
    Doxycycline Inducible Atf3 Expression, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/doxycycline inducible atf3 expression/product/Thermo Fisher
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    doxycycline inducible atf3 expression - by Bioz Stars, 2020-05
    91/100 stars
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    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

    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: 3’UTR reporter assays 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

    Regulation of GRB2 by EBV miR-BHRF1-2-5p contributes to the growth of latently infected LCLs. A. Ectopic expression of the BHRF1-2 miRNAs enhances the growth of mutant LCLs. BHRF1-2 miRNA mutant LCLs stably transduced with pLCE or pLCE-BHRF1-2 (Donor 1 = LCLBACD2; Donor 2 = LCL-D2) were plated in triplicate or quadruplicate at 2.5 x 10^6 cells per mL in media containing 10% FBS or 20% FBS (see also S3 Fig ). Viable cell counts were determined at times indicated in S3 Fig . Cell growth rates were calculated between 2 and 5 days post-plating using the equation: ln(N1/N1) = k(t1-t2), where k = growth rate, t = time, and N = cell number. *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: Regulation of GRB2 by EBV miR-BHRF1-2-5p contributes to the growth of latently infected LCLs. A. Ectopic expression of the BHRF1-2 miRNAs enhances the growth of mutant LCLs. BHRF1-2 miRNA mutant LCLs stably transduced with pLCE or pLCE-BHRF1-2 (Donor 1 = LCLBACD2; Donor 2 = LCL-D2) were plated in triplicate or quadruplicate at 2.5 x 10^6 cells per mL in media containing 10% FBS or 20% FBS (see also S3 Fig ). Viable cell counts were determined at times indicated in S3 Fig . Cell growth rates were calculated between 2 and 5 days post-plating using the equation: ln(N1/N1) = k(t1-t2), where k = growth rate, t = time, and N = cell number. *By Student’s t-test, p

    Article Snippet: 3’UTR reporter assays 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: Infection, Expressing, Mutagenesis, Stable Transfection, Transduction

    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: 3’UTR reporter assays 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: 3’UTR reporter assays 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: 3’UTR reporter assays 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. A. EBV miRNA interactions identified in PAR-CLIP datasets from EBV B95-8 or wild-type LCLs and EBV+/KSHV+ BC1 cells. CLIP’ed cellular 3’UTR sites with > = 6mer canonical seed matches (nt 2–7) to the indicated EBV miRNAs were compared to a BCR-associated gene list curated from six databases (see Methods ). 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. A. EBV miRNA interactions identified in PAR-CLIP datasets from EBV B95-8 or wild-type LCLs and EBV+/KSHV+ BC1 cells. CLIP’ed cellular 3’UTR sites with > = 6mer canonical seed matches (nt 2–7) to the indicated EBV miRNAs were compared to a BCR-associated gene list curated from six databases (see Methods ). 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: 3’UTR reporter assays 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: Cross-linking Immunoprecipitation, 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: 3’UTR reporter assays 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: 3’UTR reporter assays 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

    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

    Atf3 deletion in mouse dermal fibroblasts enhances tumorigenic behavior of keratinocyte-derived SCC. (A) RT-qPCR analysis of the indicated genes in multiple parallel cultures of primary dermal fibroblasts derived from either individual or pooled newborn Atf3 −/− ( n = 8) and Atf3 +/+ ( n = 5) mice. Values are expressed as log10 ratios in Atf3 −/− versus Atf3 +/+ cultures. Data are mean ± SEM, one-tailed one-sample t test, *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation

    doi: 10.1084/jem.20170724

    Figure Lengend Snippet: Atf3 deletion in mouse dermal fibroblasts enhances tumorigenic behavior of keratinocyte-derived SCC. (A) RT-qPCR analysis of the indicated genes in multiple parallel cultures of primary dermal fibroblasts derived from either individual or pooled newborn Atf3 −/− ( n = 8) and Atf3 +/+ ( n = 5) mice. Values are expressed as log10 ratios in Atf3 −/− versus Atf3 +/+ cultures. Data are mean ± SEM, one-tailed one-sample t test, *, P

    Article Snippet: For doxycycline-inducible Atf3 expression, the human Atf3 coding sequence was amplified from pMXs-Atf3 by PCR and cloned into a TOPO-pENTR vector, followed by transfer into pINDUCER20 ( ) using the Gateway LR recombinase system (Thermo Fisher Scientific). pMXs-Atf3 and pINDUCER20 lentivirus were packaged in HEK293T cells and incubated with HDFs for 24 h. Cells were selected, respectively, with 500 μg/ml of G418 for 7 d and 1 μg/ml of puromycin for 5 d for pMXs-Atf3 and pINDUCER20-ATF3 2 d after selection.

    Techniques: Derivative Assay, Quantitative RT-PCR, Mouse Assay, One-tailed Test

    BET inhibitors function as global suppressors of CAF gene transcription, with overlapping impact with ATF3- and CSL-dependent gene transcription. (A and B) Two independent HDF strains with Atf3 gene silencing by two siRNAs (no. 1 and 2) were treated 24 h after transfection with increasing doses of JQ1 (25, 100, and 500 nM) or DMSO vehicle for an additional 48 h. As a reference, a parallel set of cells was transfected with anti- Atf3 siRNAs versus scrambled siRNA control. Expression of the indicated CAF effector (A) and positive regulator (B) genes was assessed by RT-qPCR. Results are shown as a heat map of ratios of gene expression (folds of down- or up-regulation in blue and magenta, respectively) in drug-treated versus DMSO control HDF and in cells with and without Atf3 silencing. (C) Three independent CAF strains from surgically excised skin SCCs were treated with increasing amounts (25, 100, and 500 nM) of three different inhibitors—JQ1, OTX-015 (OTX), and iBET 762 (iBET)—in parallel with DMSO alone for 48 h followed by RT-qPCR analysis of the indicated genes shown as a heat map plot. (D) CAF strains were treated with JQ1 (25, 100, and 500 nM) or DMSO control for 48 h, followed by RT-qPCR analysis of the indicated CAF-positive regulator genes shown as a heat map plot. (E) SCC13 cells were treated with JQ1 at increasing amounts (25, 100, and 500 nM) versus DMSO vehicle alone for 48 h, followed by RT-qPCR analysis of the indicated genes and heat map plotting of the results. (F) Morphological changes induced in SCC13 cells by JQ1 treatment (500 nM) versus DMSO vehicle for 48 h. (G) Alamar blue cell density assays of SCC13 cells in triplicate wells treated with JQ1 at the indicated doses or DMSO alone, with daily measurements for 4 d. (H–J) Plot of gene set enrichment analysis (GSEA) using RNA-seq expression profile of three CAF strains with and without JQ1 treatment (500 nM for 2 d) versus vehicle (DMSO) against (H) set of genes down-modulated by induced ATF3, (I) set of genes up-regulated by siRNA-mediated Atf3 silencing, and (J) set of genes up-regulated by siRNA-mediated Csl silencing. Genes are ranked by the signal-to-noise ratio based on their differential expression in CAFs with JQ1 versus vehicle treatment; positions of the genes from the gene set are indicated by vertical bars, and the enrichment score is shown in green. The complete list of significantly enriched process networks (P

    Journal: The Journal of Experimental Medicine

    Article Title: Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation

    doi: 10.1084/jem.20170724

    Figure Lengend Snippet: BET inhibitors function as global suppressors of CAF gene transcription, with overlapping impact with ATF3- and CSL-dependent gene transcription. (A and B) Two independent HDF strains with Atf3 gene silencing by two siRNAs (no. 1 and 2) were treated 24 h after transfection with increasing doses of JQ1 (25, 100, and 500 nM) or DMSO vehicle for an additional 48 h. As a reference, a parallel set of cells was transfected with anti- Atf3 siRNAs versus scrambled siRNA control. Expression of the indicated CAF effector (A) and positive regulator (B) genes was assessed by RT-qPCR. Results are shown as a heat map of ratios of gene expression (folds of down- or up-regulation in blue and magenta, respectively) in drug-treated versus DMSO control HDF and in cells with and without Atf3 silencing. (C) Three independent CAF strains from surgically excised skin SCCs were treated with increasing amounts (25, 100, and 500 nM) of three different inhibitors—JQ1, OTX-015 (OTX), and iBET 762 (iBET)—in parallel with DMSO alone for 48 h followed by RT-qPCR analysis of the indicated genes shown as a heat map plot. (D) CAF strains were treated with JQ1 (25, 100, and 500 nM) or DMSO control for 48 h, followed by RT-qPCR analysis of the indicated CAF-positive regulator genes shown as a heat map plot. (E) SCC13 cells were treated with JQ1 at increasing amounts (25, 100, and 500 nM) versus DMSO vehicle alone for 48 h, followed by RT-qPCR analysis of the indicated genes and heat map plotting of the results. (F) Morphological changes induced in SCC13 cells by JQ1 treatment (500 nM) versus DMSO vehicle for 48 h. (G) Alamar blue cell density assays of SCC13 cells in triplicate wells treated with JQ1 at the indicated doses or DMSO alone, with daily measurements for 4 d. (H–J) Plot of gene set enrichment analysis (GSEA) using RNA-seq expression profile of three CAF strains with and without JQ1 treatment (500 nM for 2 d) versus vehicle (DMSO) against (H) set of genes down-modulated by induced ATF3, (I) set of genes up-regulated by siRNA-mediated Atf3 silencing, and (J) set of genes up-regulated by siRNA-mediated Csl silencing. Genes are ranked by the signal-to-noise ratio based on their differential expression in CAFs with JQ1 versus vehicle treatment; positions of the genes from the gene set are indicated by vertical bars, and the enrichment score is shown in green. The complete list of significantly enriched process networks (P

    Article Snippet: For doxycycline-inducible Atf3 expression, the human Atf3 coding sequence was amplified from pMXs-Atf3 by PCR and cloned into a TOPO-pENTR vector, followed by transfer into pINDUCER20 ( ) using the Gateway LR recombinase system (Thermo Fisher Scientific). pMXs-Atf3 and pINDUCER20 lentivirus were packaged in HEK293T cells and incubated with HDFs for 24 h. Cells were selected, respectively, with 500 μg/ml of G418 for 7 d and 1 μg/ml of puromycin for 5 d for pMXs-Atf3 and pINDUCER20-ATF3 2 d after selection.

    Techniques: Transfection, Expressing, Quantitative RT-PCR, RNA Sequencing Assay

    Atf3 and Csl gene silencing cause similar overlapping changes in global chromatin configuration. ChIP-seq analysis of histone marks (H3K27ac and H3K27me3) and RNA Pol II in HDFs upon siRNA-mediated silencing of Atf3 and Csl compared with the scrambled siRNA control. (A) Difference in H3K27 acetylation along the length of two representative chromosomes (CHR 1 and 7) in HDFs with silencing of Atf3 (si Atf3 ) and Csl (si Csl ), individually and in combination (si Atf3 and Csl ) relative to control cells (siCtrl). The approximated location of individual CAF-related genes and endogenous ATF3 binding position as assessed by ChIP-seq are shown below the chromosomal maps. Individual patterns of H3K27ac along the length of the two chromosomes in A are shown in Fig. S2 C. Differences in H3K27ac for the other two chromosomes (CHR 2 and 4) are shown in Fig. S2 D. (B) Changes in H3K27 acetylation and trimethylation together with Pol-II recruitment caused by Atf3 silencing at genomic loci encompassing endogenous ATF3 binding sites and flanking Tead4 and Il6 genes. Peaks of histone modification or Pol-II present in control HDFs (siCTRL) are shown in pink, peaks in HDFs with Atf3 gene silencing (si Atf3 ) are shown in turquoise, and overlapping areas present in both cells are shown in purple. For each panel, the chromosomal locations of the shown loci and scale bars are depicted at the bottom and top, respectively. Positions of ATF3 binding sites (calculated by MACS; Table S6) are indicated by vertical black arrows, and their zoom-in binding peaks are shown in Fig. S3 B. Lower lines are transcribed and spliced regions (blue boxes = coding exons) as predicted by Ensembl, together with the position and direction of transcription start sites (horizontal arrows). (C) “Zoomed in” chromatin modifications of the Tead4 and Il6 loci corresponding to the boxed region in B. Individual profiles for each condition were visualized in IGV and merged using Adobe Photoshop.

    Journal: The Journal of Experimental Medicine

    Article Title: Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation

    doi: 10.1084/jem.20170724

    Figure Lengend Snippet: Atf3 and Csl gene silencing cause similar overlapping changes in global chromatin configuration. ChIP-seq analysis of histone marks (H3K27ac and H3K27me3) and RNA Pol II in HDFs upon siRNA-mediated silencing of Atf3 and Csl compared with the scrambled siRNA control. (A) Difference in H3K27 acetylation along the length of two representative chromosomes (CHR 1 and 7) in HDFs with silencing of Atf3 (si Atf3 ) and Csl (si Csl ), individually and in combination (si Atf3 and Csl ) relative to control cells (siCtrl). The approximated location of individual CAF-related genes and endogenous ATF3 binding position as assessed by ChIP-seq are shown below the chromosomal maps. Individual patterns of H3K27ac along the length of the two chromosomes in A are shown in Fig. S2 C. Differences in H3K27ac for the other two chromosomes (CHR 2 and 4) are shown in Fig. S2 D. (B) Changes in H3K27 acetylation and trimethylation together with Pol-II recruitment caused by Atf3 silencing at genomic loci encompassing endogenous ATF3 binding sites and flanking Tead4 and Il6 genes. Peaks of histone modification or Pol-II present in control HDFs (siCTRL) are shown in pink, peaks in HDFs with Atf3 gene silencing (si Atf3 ) are shown in turquoise, and overlapping areas present in both cells are shown in purple. For each panel, the chromosomal locations of the shown loci and scale bars are depicted at the bottom and top, respectively. Positions of ATF3 binding sites (calculated by MACS; Table S6) are indicated by vertical black arrows, and their zoom-in binding peaks are shown in Fig. S3 B. Lower lines are transcribed and spliced regions (blue boxes = coding exons) as predicted by Ensembl, together with the position and direction of transcription start sites (horizontal arrows). (C) “Zoomed in” chromatin modifications of the Tead4 and Il6 loci corresponding to the boxed region in B. Individual profiles for each condition were visualized in IGV and merged using Adobe Photoshop.

    Article Snippet: For doxycycline-inducible Atf3 expression, the human Atf3 coding sequence was amplified from pMXs-Atf3 by PCR and cloned into a TOPO-pENTR vector, followed by transfer into pINDUCER20 ( ) using the Gateway LR recombinase system (Thermo Fisher Scientific). pMXs-Atf3 and pINDUCER20 lentivirus were packaged in HEK293T cells and incubated with HDFs for 24 h. Cells were selected, respectively, with 500 μg/ml of G418 for 7 d and 1 μg/ml of puromycin for 5 d for pMXs-Atf3 and pINDUCER20-ATF3 2 d after selection.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Modification, Magnetic Cell Separation

    Induced Atf3 suppresses CAF effector genes with large overlap of binding targets with CSL. (A) Several HDF strains infected with a lentiviral vector for Doxycycline (Dox)–inducible Atf3 expression (pInd- Atf3 ; n = 3) versus empty vector control (pInd-Ctr; n = 2) were analyzed with and without Dox treatment (750 ng/ml for 3 d) for expression of the indicated genes. Values are expressed as ratio log10 (pInd- Atf3 /pInd-Ctr), two-tailed, one-sample t test, *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation

    doi: 10.1084/jem.20170724

    Figure Lengend Snippet: Induced Atf3 suppresses CAF effector genes with large overlap of binding targets with CSL. (A) Several HDF strains infected with a lentiviral vector for Doxycycline (Dox)–inducible Atf3 expression (pInd- Atf3 ; n = 3) versus empty vector control (pInd-Ctr; n = 2) were analyzed with and without Dox treatment (750 ng/ml for 3 d) for expression of the indicated genes. Values are expressed as ratio log10 (pInd- Atf3 /pInd-Ctr), two-tailed, one-sample t test, *, P

    Article Snippet: For doxycycline-inducible Atf3 expression, the human Atf3 coding sequence was amplified from pMXs-Atf3 by PCR and cloned into a TOPO-pENTR vector, followed by transfer into pINDUCER20 ( ) using the Gateway LR recombinase system (Thermo Fisher Scientific). pMXs-Atf3 and pINDUCER20 lentivirus were packaged in HEK293T cells and incubated with HDFs for 24 h. Cells were selected, respectively, with 500 μg/ml of G418 for 7 d and 1 μg/ml of puromycin for 5 d for pMXs-Atf3 and pINDUCER20-ATF3 2 d after selection.

    Techniques: Binding Assay, Infection, Plasmid Preparation, Expressing, Two Tailed Test

    Overlapping transcription repressive function of low levels of ATF3 and CSL. (A) RT-qPCR analysis of CAF effector genes in several independent strains of HDFs (strain 1 tested twice at different passages) with and without Atf3 silencing by two different siRNAs (no. 1 and no. 2), using 36β4 for normalization. n (HDF strains) = 8 si Atf3 no.1 and 2; 4 control siRNA. Values in A–C are expressed as log2 ratios ( Atf3 siRNA/control siRNA), one-tailed, one-sample t test, *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation

    doi: 10.1084/jem.20170724

    Figure Lengend Snippet: Overlapping transcription repressive function of low levels of ATF3 and CSL. (A) RT-qPCR analysis of CAF effector genes in several independent strains of HDFs (strain 1 tested twice at different passages) with and without Atf3 silencing by two different siRNAs (no. 1 and no. 2), using 36β4 for normalization. n (HDF strains) = 8 si Atf3 no.1 and 2; 4 control siRNA. Values in A–C are expressed as log2 ratios ( Atf3 siRNA/control siRNA), one-tailed, one-sample t test, *, P

    Article Snippet: For doxycycline-inducible Atf3 expression, the human Atf3 coding sequence was amplified from pMXs-Atf3 by PCR and cloned into a TOPO-pENTR vector, followed by transfer into pINDUCER20 ( ) using the Gateway LR recombinase system (Thermo Fisher Scientific). pMXs-Atf3 and pINDUCER20 lentivirus were packaged in HEK293T cells and incubated with HDFs for 24 h. Cells were selected, respectively, with 500 μg/ml of G418 for 7 d and 1 μg/ml of puromycin for 5 d for pMXs-Atf3 and pINDUCER20-ATF3 2 d after selection.

    Techniques: Quantitative RT-PCR, One-tailed Test

    Atf3 deficiency promotes dysplastic keratinocyte tumor development and widespread stromal alterations. (A) Laser-captured microdissection (LCM) and RT-qPCR analysis of Atf3 expression in actinic keratosis (AK)–underlying stromas ( n = 7) and matched flanking normal skin stromas (normal; n = 7) from patients using 36β4 normalization. Data are mean ± SEM, two-tailed paired t test, *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation

    doi: 10.1084/jem.20170724

    Figure Lengend Snippet: Atf3 deficiency promotes dysplastic keratinocyte tumor development and widespread stromal alterations. (A) Laser-captured microdissection (LCM) and RT-qPCR analysis of Atf3 expression in actinic keratosis (AK)–underlying stromas ( n = 7) and matched flanking normal skin stromas (normal; n = 7) from patients using 36β4 normalization. Data are mean ± SEM, two-tailed paired t test, *, P

    Article Snippet: For doxycycline-inducible Atf3 expression, the human Atf3 coding sequence was amplified from pMXs-Atf3 by PCR and cloned into a TOPO-pENTR vector, followed by transfer into pINDUCER20 ( ) using the Gateway LR recombinase system (Thermo Fisher Scientific). pMXs-Atf3 and pINDUCER20 lentivirus were packaged in HEK293T cells and incubated with HDFs for 24 h. Cells were selected, respectively, with 500 μg/ml of G418 for 7 d and 1 μg/ml of puromycin for 5 d for pMXs-Atf3 and pINDUCER20-ATF3 2 d after selection.

    Techniques: Laser Capture Microdissection, Quantitative RT-PCR, Expressing, Two Tailed Test

    Deletion of an ATF3 binding region 2 Mb upstream of the IL6 gene results in specific induction of IL6 expression . (A) Overview of the construction of a dual-guide RNA (gRNA)–Cas9 expression vector. A two-step PCR amplification followed by insertion into the lentiCRISPR_v2 vector backbone was used to create a dual-expression cassette with mouse (mU6) and human (hU6) U6 promoters driving expression of two gRNAs targeting the ATF3 binding region upstream of the Il6 locus. For details, see Materials and methods. (B, top) Schematic representation of ATF3 binding region 2.07 Mb upstream of the Il6 locus, with the position of the dual gRNAs (gRNA 1 and 2) chosen for CRISPR/Cas9–mediated deletion and of the two primers (P1 and P2) used for genomic PCR analysis. (B, bottom) Nucleotide sequence of the genomic locus targeted by the two gRNAs (blue and red, respectively) together with PAM motifs (bold lines); below are the nucleotide-sequencing results of 300-bp genomic PCR products derived from two HDF clones expected to harbor the deletion (as shown in the panel below). (C) Small clusters/colonies of HDFs infected with the dual gRNAs lentiCRISPR vector (▵) and empty vector control (C) were analyzed by genomic PCR analysis with the P1 and P2 primers indicated above. Clones harboring the deletion in either a heterozygous or homozygous state, on the basis of PCR products of 300 bp versus 500 bp in size, were further analyzed by RT-qPCR for levels of Il6 , Tnc , and Tead4 expression. (D) Two additional deletion-harboring clones were analyzed together with controls by IF with Ab against IL6 and TNC. Shown are representative images together with a quantification of the fluorescence intensity signal in individual cells of deletion-harboring clones (▵) versus controls (C). n = 20 cells per condition, mean ± SEM, two-tailed unpaired t test, *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Convergent roles of ATF3 and CSL in chromatin control of cancer-associated fibroblast activation

    doi: 10.1084/jem.20170724

    Figure Lengend Snippet: Deletion of an ATF3 binding region 2 Mb upstream of the IL6 gene results in specific induction of IL6 expression . (A) Overview of the construction of a dual-guide RNA (gRNA)–Cas9 expression vector. A two-step PCR amplification followed by insertion into the lentiCRISPR_v2 vector backbone was used to create a dual-expression cassette with mouse (mU6) and human (hU6) U6 promoters driving expression of two gRNAs targeting the ATF3 binding region upstream of the Il6 locus. For details, see Materials and methods. (B, top) Schematic representation of ATF3 binding region 2.07 Mb upstream of the Il6 locus, with the position of the dual gRNAs (gRNA 1 and 2) chosen for CRISPR/Cas9–mediated deletion and of the two primers (P1 and P2) used for genomic PCR analysis. (B, bottom) Nucleotide sequence of the genomic locus targeted by the two gRNAs (blue and red, respectively) together with PAM motifs (bold lines); below are the nucleotide-sequencing results of 300-bp genomic PCR products derived from two HDF clones expected to harbor the deletion (as shown in the panel below). (C) Small clusters/colonies of HDFs infected with the dual gRNAs lentiCRISPR vector (▵) and empty vector control (C) were analyzed by genomic PCR analysis with the P1 and P2 primers indicated above. Clones harboring the deletion in either a heterozygous or homozygous state, on the basis of PCR products of 300 bp versus 500 bp in size, were further analyzed by RT-qPCR for levels of Il6 , Tnc , and Tead4 expression. (D) Two additional deletion-harboring clones were analyzed together with controls by IF with Ab against IL6 and TNC. Shown are representative images together with a quantification of the fluorescence intensity signal in individual cells of deletion-harboring clones (▵) versus controls (C). n = 20 cells per condition, mean ± SEM, two-tailed unpaired t test, *, P

    Article Snippet: For doxycycline-inducible Atf3 expression, the human Atf3 coding sequence was amplified from pMXs-Atf3 by PCR and cloned into a TOPO-pENTR vector, followed by transfer into pINDUCER20 ( ) using the Gateway LR recombinase system (Thermo Fisher Scientific). pMXs-Atf3 and pINDUCER20 lentivirus were packaged in HEK293T cells and incubated with HDFs for 24 h. Cells were selected, respectively, with 500 μg/ml of G418 for 7 d and 1 μg/ml of puromycin for 5 d for pMXs-Atf3 and pINDUCER20-ATF3 2 d after selection.

    Techniques: Binding Assay, Expressing, Plasmid Preparation, Polymerase Chain Reaction, Amplification, CRISPR, Sequencing, Derivative Assay, Clone Assay, Infection, Quantitative RT-PCR, Fluorescence, Two Tailed Test