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    Name:
    5 Ethynyl 2 deoxyuridine
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    clk-n001-100
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    Structured Review

    Jena Bioscience edu
    Expression kinetics and subcellular localization of HCMV lncRNAs. a) Expression levels of HCMV encoded lncRNAs (RNA2.7, RNA1.2, RNA4.9 and exonic RNA5.0) together with the median expression of viral genes and one host transcript (ACTB) as measured by RNA-seq during HCMV infection in fibroblasts (MOI = 5) [ 24 ]. b) HCMV lncRNAs were detected by RNA-FISH using fluorescent probes (white) in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 1). c) RNA4.9 and the UL44 protein were detected in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 5) using RNA-FISH and IF, respectively. Differential interference contrast (DIC) of the stained cell shows viral <t>DNA</t> replication compartments, indicated by black arrows. d) RNA4.9 and nascent DNA were detected in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 3) using RNA-FISH and <t>EdU</t> incorporation followed by labelling with a 6-FAM fluorescent azide using the “Click” chemistry. b-d) Nuclei were counterstained with Hoechst (blue, in merge).

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    1) Product Images from "Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication"

    Article Title: Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008390

    Expression kinetics and subcellular localization of HCMV lncRNAs. a) Expression levels of HCMV encoded lncRNAs (RNA2.7, RNA1.2, RNA4.9 and exonic RNA5.0) together with the median expression of viral genes and one host transcript (ACTB) as measured by RNA-seq during HCMV infection in fibroblasts (MOI = 5) [ 24 ]. b) HCMV lncRNAs were detected by RNA-FISH using fluorescent probes (white) in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 1). c) RNA4.9 and the UL44 protein were detected in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 5) using RNA-FISH and IF, respectively. Differential interference contrast (DIC) of the stained cell shows viral DNA replication compartments, indicated by black arrows. d) RNA4.9 and nascent DNA were detected in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 3) using RNA-FISH and EdU incorporation followed by labelling with a 6-FAM fluorescent azide using the “Click” chemistry. b-d) Nuclei were counterstained with Hoechst (blue, in merge).
    Figure Legend Snippet: Expression kinetics and subcellular localization of HCMV lncRNAs. a) Expression levels of HCMV encoded lncRNAs (RNA2.7, RNA1.2, RNA4.9 and exonic RNA5.0) together with the median expression of viral genes and one host transcript (ACTB) as measured by RNA-seq during HCMV infection in fibroblasts (MOI = 5) [ 24 ]. b) HCMV lncRNAs were detected by RNA-FISH using fluorescent probes (white) in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 1). c) RNA4.9 and the UL44 protein were detected in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 5) using RNA-FISH and IF, respectively. Differential interference contrast (DIC) of the stained cell shows viral DNA replication compartments, indicated by black arrows. d) RNA4.9 and nascent DNA were detected in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 3) using RNA-FISH and EdU incorporation followed by labelling with a 6-FAM fluorescent azide using the “Click” chemistry. b-d) Nuclei were counterstained with Hoechst (blue, in merge).

    Techniques Used: Expressing, RNA Sequencing Assay, Infection, Fluorescence In Situ Hybridization, Staining

    Related Articles

    Labeling:

    Article Title: Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication.
    Article Snippet: Immunofluorescence, EdU staining and FISH Cells were plated on μ-Slide 8 well (ibidi Gmbh) chambers infected as indicated in the figure legends, washed once with PBS and fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature (RT). .. After fixation, the cells were washed twice with PBS and permeabilized with 0.5% (v/v) Triton X-100 in PBS for 20 min. For metabolic labeling of nascent DNA, EdU (Jena Bioscience GmbH) was added to the culture medium at 10 μM and incubated for 30 min prior to fixation. ..

    Article Title: WAPL-Dependent Repair of Damaged DNA Replication Forks Underlies Oncogene-Induced Loss of Sister Chromatid Cohesion.
    Article Snippet: For FUCCI time-lapse experiment, images were taken using a 20x objective with 1.6 optovar every 30 min. For the H2B-YFP time-lapse experiment, images were a taken using a 20x objective every 7 min. Mitotic duration in RPE1 cells was assessed by adding 1 mM siR-DNA (Spirochrome) to stain DNA and imaging every 5 min with an IncuCyte Zoom instrument (Essen Bioscience) using a 20x objective. .. Flow Cytometry Cells were labeled with 10 mM 5’-ethynyl-2’-deoxyuridine (EdU, Jena Bioscience) for 10 min, harvested and washed with 1% BSA. .. For fixation, cells were first resuspended in 4% paraformaldehyde for 15 min at room temperature, then centrifuged and stored in 70% EtOH at -20 C. For mitosis detection, cells were washed in 1% BSA, permeabilized in 0.5% Triton X-100, blocked with 5% FCS for 1h and incubated with Alexa Fluor 647 anti-Histone H3 pS10 (clone 11d 8, Biolegend) for 2h.

    Article Title: Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication
    Article Snippet: Immunofluorescence, EdU staining and FISHCells were plated on μ-Slide 8 well chambers (ibidi Gmbh) infected as indicated in the figure legends, washed once with PBS and fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature (RT). .. After fixation, the cells were washed twice with PBS and permeabilized with 0.5% (v/v) Triton X-100 in PBS for 20 min. For metabolic labeling of nascent DNA, EdU (Jena Bioscience GmbH) was added to the culture medium at 10 μM and incubated for 30 min prior to fixation. ..

    Incubation:

    Article Title: Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication.
    Article Snippet: Immunofluorescence, EdU staining and FISH Cells were plated on μ-Slide 8 well (ibidi Gmbh) chambers infected as indicated in the figure legends, washed once with PBS and fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature (RT). .. After fixation, the cells were washed twice with PBS and permeabilized with 0.5% (v/v) Triton X-100 in PBS for 20 min. For metabolic labeling of nascent DNA, EdU (Jena Bioscience GmbH) was added to the culture medium at 10 μM and incubated for 30 min prior to fixation. ..

    Article Title: Rho-Associated Coiled-Coil Kinase 1 Translocates to the Nucleus and Inhibits Human Cytomegalovirus Propagation
    Article Snippet: EdU staining was performed based on the method in reference . .. Briefly, cells were incubated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) (Jena Bioscience GmbH) for 30 min. .. Cells were then fixed with 4% formaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 20 min, and stained with staining mix (100 mM Tris, pH 8.5, 1 mM CuSO4 , 10 μM fluorescent azide, 100 mM ascorbic acid) for 30 min. EdU-stained cells were immunostained for ROCK1 and DAPI as described above.

    Article Title: Chromatin Trapping of Factors Involved in DNA Replication and Repair Underlies Heat-Induced Radio- and Chemosensitization
    Article Snippet: The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; PanEco, Moscow, Russia) supplemented with 10% fetal bovine serum (FBS; GE Healthcare) at 37 °C in a humidified CO2 incubator. .. For hyperthermia, cells were immersed in a precision-controlled water bath at 42–45 °C (±0.05 °C) for 30 min. To induce double-stranded DNA breaks (DSBs), cells were treated with 20 µg/mL etoposide (Sigma-Aldrich, St. Louis, MO, USA) for 1 h; to induce single-stranded DNA breaks (SSBs), cells were treated with 200 µM peroxide hydrogen (Sigma-Aldrich) for 1 h; to induce replication stress, cells were treated with 10 mM hydroxyurea (Sigma-Aldrich) or with 10 µM aphidicolin (Sigma-Aldrich) for 1 h; for inhibition of lagging strand synthesis, cells were treated with 2 mM emetine (Sigma-Aldrich) for 1 h; for c-trapping induction, cells were treated with 10 µM curaxin CBL0137 (Selleckchem, Houston, TX, USA) for 1 h. To label active replication sites, cells were incubated with 10 μM 5-ethynyl-2’-deoxyuridine (EdU; Jena Biosciences, Jena, Germany) for 20 min at 37 °C. .. Incorporated EdU were visualized using a Click−iT EdU Imaging Kit (Invitrogen) according to the manufacturer’s instructions.

    Article Title: Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication
    Article Snippet: Immunofluorescence, EdU staining and FISHCells were plated on μ-Slide 8 well chambers (ibidi Gmbh) infected as indicated in the figure legends, washed once with PBS and fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature (RT). .. After fixation, the cells were washed twice with PBS and permeabilized with 0.5% (v/v) Triton X-100 in PBS for 20 min. For metabolic labeling of nascent DNA, EdU (Jena Bioscience GmbH) was added to the culture medium at 10 μM and incubated for 30 min prior to fixation. ..

    Flow Cytometry:

    Article Title: WAPL-Dependent Repair of Damaged DNA Replication Forks Underlies Oncogene-Induced Loss of Sister Chromatid Cohesion.
    Article Snippet: For FUCCI time-lapse experiment, images were taken using a 20x objective with 1.6 optovar every 30 min. For the H2B-YFP time-lapse experiment, images were a taken using a 20x objective every 7 min. Mitotic duration in RPE1 cells was assessed by adding 1 mM siR-DNA (Spirochrome) to stain DNA and imaging every 5 min with an IncuCyte Zoom instrument (Essen Bioscience) using a 20x objective. .. Flow Cytometry Cells were labeled with 10 mM 5’-ethynyl-2’-deoxyuridine (EdU, Jena Bioscience) for 10 min, harvested and washed with 1% BSA. .. For fixation, cells were first resuspended in 4% paraformaldehyde for 15 min at room temperature, then centrifuged and stored in 70% EtOH at -20 C. For mitosis detection, cells were washed in 1% BSA, permeabilized in 0.5% Triton X-100, blocked with 5% FCS for 1h and incubated with Alexa Fluor 647 anti-Histone H3 pS10 (clone 11d 8, Biolegend) for 2h.

    In Vivo:

    Article Title: PDGFRα signaling drives adipose tissue fibrosis by targeting progenitor cell plasticity
    Article Snippet: .. For in vivo EdU experiments, 2 mM EdU (Jena Bioscience) in 200 µL of 0.9% saline was injected intraperitoneally twice at 24 and 8 h before analysis. .. To study Nestin-Cre/Tomato labeling of adipocytes, some mice were fed a HFD (60% calories from fat) (Harlan Teklad, #TD06414) for up to 12 wk.

    Injection:

    Article Title: PDGFRα signaling drives adipose tissue fibrosis by targeting progenitor cell plasticity
    Article Snippet: .. For in vivo EdU experiments, 2 mM EdU (Jena Bioscience) in 200 µL of 0.9% saline was injected intraperitoneally twice at 24 and 8 h before analysis. .. To study Nestin-Cre/Tomato labeling of adipocytes, some mice were fed a HFD (60% calories from fat) (Harlan Teklad, #TD06414) for up to 12 wk.

    Article Title: Nonlinear ionizing radiation-induced changes in eye lens cell proliferation, cyclin D1 expression and lens shape
    Article Snippet: Animal irradiation studies Six-week-old C57BL/6J mice (Harlan, UK), in groups of two males and two females, were exposed to single doses of IR in an X-ray chamber irradiator (250 kVp, with Gulway generator (AGO Ltd, model no.: CD160/1 Serial no.: 1032–1109; copper- and aluminium-filtered 250kVp X-rays; dose rates of 5 mGy min−1 for doses up to 250 mGy and 500 mGy min−1 for the 100 and 250, 1000 and 2000 mGy dose points; both dose rates for 100 and 250 mGy). .. Each animal received a single intraperitoneal injection of EdU (Jena Bioscience GmbH, Germany) at a dose of 90 mg kg−1 body weight, 1 h before irradiation. .. All procedures strictly followed the UK Animals (Scientific Procedures) Act 1986 and had ethical approval of the UK Home Office and local AWERB (Animal Welfare and Ethical Review Body) Committee.

    Inhibition:

    Article Title: Chromatin Trapping of Factors Involved in DNA Replication and Repair Underlies Heat-Induced Radio- and Chemosensitization
    Article Snippet: The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; PanEco, Moscow, Russia) supplemented with 10% fetal bovine serum (FBS; GE Healthcare) at 37 °C in a humidified CO2 incubator. .. For hyperthermia, cells were immersed in a precision-controlled water bath at 42–45 °C (±0.05 °C) for 30 min. To induce double-stranded DNA breaks (DSBs), cells were treated with 20 µg/mL etoposide (Sigma-Aldrich, St. Louis, MO, USA) for 1 h; to induce single-stranded DNA breaks (SSBs), cells were treated with 200 µM peroxide hydrogen (Sigma-Aldrich) for 1 h; to induce replication stress, cells were treated with 10 mM hydroxyurea (Sigma-Aldrich) or with 10 µM aphidicolin (Sigma-Aldrich) for 1 h; for inhibition of lagging strand synthesis, cells were treated with 2 mM emetine (Sigma-Aldrich) for 1 h; for c-trapping induction, cells were treated with 10 µM curaxin CBL0137 (Selleckchem, Houston, TX, USA) for 1 h. To label active replication sites, cells were incubated with 10 μM 5-ethynyl-2’-deoxyuridine (EdU; Jena Biosciences, Jena, Germany) for 20 min at 37 °C. .. Incorporated EdU were visualized using a Click−iT EdU Imaging Kit (Invitrogen) according to the manufacturer’s instructions.

    Irradiation:

    Article Title: Nonlinear ionizing radiation-induced changes in eye lens cell proliferation, cyclin D1 expression and lens shape
    Article Snippet: Animal irradiation studies Six-week-old C57BL/6J mice (Harlan, UK), in groups of two males and two females, were exposed to single doses of IR in an X-ray chamber irradiator (250 kVp, with Gulway generator (AGO Ltd, model no.: CD160/1 Serial no.: 1032–1109; copper- and aluminium-filtered 250kVp X-rays; dose rates of 5 mGy min−1 for doses up to 250 mGy and 500 mGy min−1 for the 100 and 250, 1000 and 2000 mGy dose points; both dose rates for 100 and 250 mGy). .. Each animal received a single intraperitoneal injection of EdU (Jena Bioscience GmbH, Germany) at a dose of 90 mg kg−1 body weight, 1 h before irradiation. .. All procedures strictly followed the UK Animals (Scientific Procedures) Act 1986 and had ethical approval of the UK Home Office and local AWERB (Animal Welfare and Ethical Review Body) Committee.

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    • 5 ethynyl 2 deoxyuridine edu  (Jena Bioscience)
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    Jena Bioscience 5 ethynyl 2 deoxyuridine edu
    Nuclear ROCK1 colocalizes with Hsc70 at nucleolus. (A and B) Fibroblasts were infected with the Merlin strain and stained at 72 hpi for ROCK1 (red) and DAPI (blue). Replication compartments were imaged either by metabolically labeling nascent DNA with <t>ethynyl-2′-deoxyuridine</t> <t>(EdU)</t> followed by click-mediated fluorescent labeling of EdU (green) (A) or by staining for UL57 (green) (B). (C) Fibroblasts were mock infected and stained for Hsc70 (green) and DAPI (blue) or infected with the Merlin strain and stained at 72 hpi for ROCK1 (red), Hsc70 (green), pp28 (violet), and DAPI (blue). (D) Fibroblasts were infected with the Merlin strain and stained at 72 hpi for ROCK1 (red), Hsc70 (green), the nucleolar marker fibrillarin (violet), and DAPI (blue). Scale bars are 10 μm.
    5 Ethynyl 2 Deoxyuridine Edu, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/5 ethynyl 2 deoxyuridine edu/product/Jena Bioscience
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    Nuclear ROCK1 colocalizes with Hsc70 at nucleolus. (A and B) Fibroblasts were infected with the Merlin strain and stained at 72 hpi for ROCK1 (red) and DAPI (blue). Replication compartments were imaged either by metabolically labeling nascent DNA with ethynyl-2′-deoxyuridine (EdU) followed by click-mediated fluorescent labeling of EdU (green) (A) or by staining for UL57 (green) (B). (C) Fibroblasts were mock infected and stained for Hsc70 (green) and DAPI (blue) or infected with the Merlin strain and stained at 72 hpi for ROCK1 (red), Hsc70 (green), pp28 (violet), and DAPI (blue). (D) Fibroblasts were infected with the Merlin strain and stained at 72 hpi for ROCK1 (red), Hsc70 (green), the nucleolar marker fibrillarin (violet), and DAPI (blue). Scale bars are 10 μm.

    Journal: Journal of Virology

    Article Title: Rho-Associated Coiled-Coil Kinase 1 Translocates to the Nucleus and Inhibits Human Cytomegalovirus Propagation

    doi: 10.1128/JVI.00453-19

    Figure Lengend Snippet: Nuclear ROCK1 colocalizes with Hsc70 at nucleolus. (A and B) Fibroblasts were infected with the Merlin strain and stained at 72 hpi for ROCK1 (red) and DAPI (blue). Replication compartments were imaged either by metabolically labeling nascent DNA with ethynyl-2′-deoxyuridine (EdU) followed by click-mediated fluorescent labeling of EdU (green) (A) or by staining for UL57 (green) (B). (C) Fibroblasts were mock infected and stained for Hsc70 (green) and DAPI (blue) or infected with the Merlin strain and stained at 72 hpi for ROCK1 (red), Hsc70 (green), pp28 (violet), and DAPI (blue). (D) Fibroblasts were infected with the Merlin strain and stained at 72 hpi for ROCK1 (red), Hsc70 (green), the nucleolar marker fibrillarin (violet), and DAPI (blue). Scale bars are 10 μm.

    Article Snippet: Briefly, cells were incubated with 10 μM 5-ethynyl-2′-deoxyuridine (EdU) (Jena Bioscience GmbH) for 30 min.

    Techniques: Infection, Staining, Metabolic Labelling, Labeling, Marker

    Hyperthermia induces DNA replication-associated poly(ADP-ribosyl)ation (PARylation). ( A ) The network of proteins involved in the maturation of Okazaki fragments in human cells. Proteins identified as c -trapped in response to hyperthermia are shown in green ellipses. ( B ) HEK293 cells were subjected to hyperthermia (45 °C, 30 min) and stained with antibodies against PAR. Control represents HEK293 cells that were not exposed to hyperthermia. The DNA was stained with 4,6-diamino-2-phenylindole (DAPI) (blue). Epifluorescence microscopy analysis was performed. Scale bar: 40 µm. ( C ) HEK293 cells were pulse-labeled with 5-ethynyl-2’-deoxyuridine (EdU) (10 µM, 30 min), subjected to hyperthermia (45 °C, 30 min) and stained with antibodies against PAR. Control represents untreated HEK293 cells. EdU was revealed by Click Chemistry; the DNA was stained with DAPI. Structured illumination microscopy (SIM) analysis was performed. Scale bar: 5 µm.

    Journal: Cells

    Article Title: Chromatin Trapping of Factors Involved in DNA Replication and Repair Underlies Heat-Induced Radio- and Chemosensitization

    doi: 10.3390/cells9061423

    Figure Lengend Snippet: Hyperthermia induces DNA replication-associated poly(ADP-ribosyl)ation (PARylation). ( A ) The network of proteins involved in the maturation of Okazaki fragments in human cells. Proteins identified as c -trapped in response to hyperthermia are shown in green ellipses. ( B ) HEK293 cells were subjected to hyperthermia (45 °C, 30 min) and stained with antibodies against PAR. Control represents HEK293 cells that were not exposed to hyperthermia. The DNA was stained with 4,6-diamino-2-phenylindole (DAPI) (blue). Epifluorescence microscopy analysis was performed. Scale bar: 40 µm. ( C ) HEK293 cells were pulse-labeled with 5-ethynyl-2’-deoxyuridine (EdU) (10 µM, 30 min), subjected to hyperthermia (45 °C, 30 min) and stained with antibodies against PAR. Control represents untreated HEK293 cells. EdU was revealed by Click Chemistry; the DNA was stained with DAPI. Structured illumination microscopy (SIM) analysis was performed. Scale bar: 5 µm.

    Article Snippet: For hyperthermia, cells were immersed in a precision-controlled water bath at 42–45 °C (±0.05 °C) for 30 min. To induce double-stranded DNA breaks (DSBs), cells were treated with 20 µg/mL etoposide (Sigma-Aldrich, St. Louis, MO, USA) for 1 h; to induce single-stranded DNA breaks (SSBs), cells were treated with 200 µM peroxide hydrogen (Sigma-Aldrich) for 1 h; to induce replication stress, cells were treated with 10 mM hydroxyurea (Sigma-Aldrich) or with 10 µM aphidicolin (Sigma-Aldrich) for 1 h; for inhibition of lagging strand synthesis, cells were treated with 2 mM emetine (Sigma-Aldrich) for 1 h; for c-trapping induction, cells were treated with 10 µM curaxin CBL0137 (Selleckchem, Houston, TX, USA) for 1 h. To label active replication sites, cells were incubated with 10 μM 5-ethynyl-2’-deoxyuridine (EdU; Jena Biosciences, Jena, Germany) for 20 min at 37 °C.

    Techniques: Staining, Epifluorescence Microscopy, Labeling, Microscopy

    Expression kinetics and subcellular localization of HCMV lncRNAs. a) Expression levels of HCMV encoded lncRNAs (RNA2.7, RNA1.2, RNA4.9 and exonic RNA5.0) together with the median expression of viral genes and one host transcript (ACTB) as measured by RNA-seq during HCMV infection in fibroblasts (MOI = 5) [ 24 ]. b) HCMV lncRNAs were detected by RNA-FISH using fluorescent probes (white) in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 1). c) RNA4.9 and the UL44 protein were detected in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 5) using RNA-FISH and IF, respectively. Differential interference contrast (DIC) of the stained cell shows viral DNA replication compartments, indicated by black arrows. d) RNA4.9 and nascent DNA were detected in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 3) using RNA-FISH and EdU incorporation followed by labelling with a 6-FAM fluorescent azide using the “Click” chemistry. b-d) Nuclei were counterstained with Hoechst (blue, in merge).

    Journal: PLoS Pathogens

    Article Title: Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication

    doi: 10.1371/journal.ppat.1008390

    Figure Lengend Snippet: Expression kinetics and subcellular localization of HCMV lncRNAs. a) Expression levels of HCMV encoded lncRNAs (RNA2.7, RNA1.2, RNA4.9 and exonic RNA5.0) together with the median expression of viral genes and one host transcript (ACTB) as measured by RNA-seq during HCMV infection in fibroblasts (MOI = 5) [ 24 ]. b) HCMV lncRNAs were detected by RNA-FISH using fluorescent probes (white) in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 1). c) RNA4.9 and the UL44 protein were detected in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 5) using RNA-FISH and IF, respectively. Differential interference contrast (DIC) of the stained cell shows viral DNA replication compartments, indicated by black arrows. d) RNA4.9 and nascent DNA were detected in HCMV Merlin strain-infected fibroblasts at 48 hpi (MOI = 3) using RNA-FISH and EdU incorporation followed by labelling with a 6-FAM fluorescent azide using the “Click” chemistry. b-d) Nuclei were counterstained with Hoechst (blue, in merge).

    Article Snippet: After fixation, the cells were washed twice with PBS and permeabilized with 0.5% (v/v) Triton X-100 in PBS for 20 min. For metabolic labeling of nascent DNA, EdU (Jena Bioscience GmbH) was added to the culture medium at 10 μM and incubated for 30 min prior to fixation.

    Techniques: Expressing, RNA Sequencing Assay, Infection, Fluorescence In Situ Hybridization, Staining