Journal:

Article Title: Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

doi: 10.1128/JVI.75.13.6095-6106.2001

Figure Lengend Snippet: Effect of NS5A expression on IFN signal transduction. (A) Tetracycline (Tc)-regulated expression of full-length and mutant NS5A proteins. FL-NR denotes a full-length NS5A construct derived from an IFN-nonresponsive patient, while FL-CR denotes a full-length NS5A construct derived from an IFN-responsive patient. ΔN-110, ΔN-222, ΔN-334, ΔC-117, and ΔC-230 represent NS5A deletion mutants lacking 110, 222, 334, amino acids from the amino terminus and 117 and 230 amino acids from the carboxy terminus, respectively. Protein positions are shown by arrows. Western blots were probed with HCV-infected patient serum as described in Materials and Methods. (B) Effect of NS5A expression on ISGF-3 induction. Plasmids were transiently transfected into HeLa Tet-Off cells, grown in the absence and presence of tetracycline to induce and repress NS5A, respectively, and treated with IFN to induce ISGF-3. Protein extracts were hybridized to a 32P-labeled oligonucleotide corresponding to the consensus ISRE. “puc” represents a control transfection with the pTRE-puc parental plasmid with no insert. “E3L,” “PKR,” and “E1A” represent expression of the E3L, PKR, and E1A proteins, respectively. The p48 monoclonal antibody used to form supershifts (denoted by “ss” and an arrow) is denoted by “p48 Mab”. “n” and “c” represent nuclear and cytoplasmic extracts, respectively. ISGF-3 is induced only with IFN treatment and is indicated with arrows.

Article Snippet: Human HeLa Tet-Off cells were obtained from Clontech (Palo Alto, Calif.).

Techniques: Expressing, Transduction, Mutagenesis, Construct, Derivative Assay, Western Blot, Infection, Transfection, Labeling, Plasmid Preparation

Journal:

Article Title: Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

doi: 10.1128/JVI.75.13.6095-6106.2001

Figure Lengend Snippet: NS5A expression is associated with increased IL-8 protein production and inhibition of the antiviral effects of IFN. (A) Correlation among levels of IL-8 in culture supernatants, amounts of NS5A protein expression, and levels of EMCV rescue in the trans rescue assay. HeLa cells expressing FL-NS5A-NR were grown for 48 h in medium containing 0, 0.001, 0.01, and 1.0 μg of tetracycline per ml for 48 h, treated with 20 U of IFN per ml for 24 h, and infected with EMCV at an MOI of 0.01 for 24 h. The amount of IL-8 protein in culture supernatants, determined by ELISA, is indicated in the bar graph. Changes in EMCV titers were determined 24 h postinfection by viral plaque assay. The levels of NS5A protein expression were determined by Western blot analysis of equal amounts of total cellular protein extracts and are presented as +++, ++, +, and −, which correspond to high, intermediate, low, and undetectable levels of NS5A protein, respectively, as determined by computerized scanning of the chemiluminescent signal. Fold EMCV rescue represents the difference in EMCV titers in the presence of IFN among cells treated with 0, 0.001, and 0.01 μg of tetracycline per ml versus the 1.0-μg/ml concentration. (B) IL-8 inhibits the antiviral actions of IFN in vitro. HeLa Tet-Off cells were pretreated with or without 33 ng of recombinant human IL-8 per ml for 6 h and then with 20 U of IFN per ml for 18 h. Cells were then infected with EMCV at an MOI of 0.1 for 24 h. Supernatants were harvested, and the amount of EMCV was determined by titration on L929 cells. Error bars represent standard deviations, and P values derived from Student's t tests are indicated.

Article Snippet: Human HeLa Tet-Off cells were obtained from Clontech (Palo Alto, Calif.).

Techniques: Expressing, Inhibition, Rescue Assay, Infection, Enzyme-linked Immunosorbent Assay, Viral Plaque Assay, Western Blot, Concentration Assay, In Vitro, Recombinant, Titration, Derivative Assay

Journal:

Article Title: Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

doi: 10.1128/JVI.75.13.6095-6106.2001

Figure Lengend Snippet: NS5A transactivates the IL-8 promoter. (A) Effect of NS5A expression on luciferase expression under the control of the 546-luc IL-8 promoter. The 546-luc plasmid and pTRE-FL-NS5A-NR (10 μg each) were transfected into HeLa Tet-Off cells, and cells were split in triplicate and grown in media with or without tetracycline for 48 h. Cells were then treated or not treated with IFN for 24 h. The amount of luciferase activity in cell lysates was determined using a luminometer. (B) Effect of NS5A expression on luciferase activity under the control of truncated IL-8 promoters. Ten micrograms of the relevant reporter plasmid was transfected into HeLa FL-NS5A-NR cells, and cells were grown in media with or without tetracycline for 48 h. The amount of luciferase activity in cell lysates was determined using a luminometer. 546-luc, 133-luc, and 98-luc contain 546, 133, and 98 bases of the IL-8 promoter, respectively. Error bars represent standard deviations, and P values derived from Student's t tests are indicated.

Article Snippet: Human HeLa Tet-Off cells were obtained from Clontech (Palo Alto, Calif.).

Techniques: Expressing, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Derivative Assay

Journal:

Article Title: Hepatitis C Virus Nonstructural 5A Protein Induces Interleukin-8, Leading to Partial Inhibition of the Interferon-Induced Antiviral Response

doi: 10.1128/JVI.75.13.6095-6106.2001

Figure Lengend Snippet: Characterization of domains on NS5A and the IL-8 promoter required for transactivation. (A) Effect of NS5A mutant proteins on luciferase expression under the control of the full-length (546-luc) IL-8 promoter. Ten-microgram quantities of the 546-luc plasmid and pTRE-FL-NS5A-NR, NS5A-ΔN110, NS5A-ΔN222, or NS5A-ΔC117 were transfected into HeLa Tet-Off cells, and cells were split in triplicate and grown in media with or without tetracycline for 48 h. The amount of luciferase activity in cell lysates was determined using a luminometer. The data shown are the fold changes in luciferase activity with the gene in the induced versus the uninduced state. (B) Im- munofluoresence analysis of HeLa cells expressing FL-NS5A-NR, NS5A-ΔN222, or NS5A-ΔC117. HeLa Tet-Off cells stably transfected with FL-NS5A-NR (top panels), NS5A-ΔN222 (middle panels), or NS5A-ΔC117 (lower panels) were grown in the absence or presence of tetracycline (Tc) to induce or repress NS5A expression for 48 h. Cells were fixed and stained with a monoclonal NS5A antibody and then with Cy3-linked anti-mouse secondary antibodies, as described in Materials and Methods. Images are at a ×100 amplification. (C) Effect of NS5A-ΔN222 expression on luciferase activity under the control of mutated IL-8 promoters. Ten-microgram quantities of NS5A-ΔN222 and the relevant reporter plasmid were cotransfected into HeLa Tet-Off cells; cells were split in triplicate and grown in media with or without tetracycline for 48 h. Cells were then treated with 4 ng of TNF-α for 6 h. The amount of luciferase activity in cell lysates was determined using a luminometer. 546-luc, 133-luc, and 98-luc contain 546, 133, and 98 bases of the IL-8 promoter, respectively. NF-κB–luc, AP-1–luc, and NF–IL-6–luc represent the 133-luc plasmid with point mutations in the NF-κB, AP-1, and NF–IL-6 binding sites.

Article Snippet: Human HeLa Tet-Off cells were obtained from Clontech (Palo Alto, Calif.).

Techniques: Mutagenesis, Luciferase, Expressing, Plasmid Preparation, Transfection, Activity Assay, Stable Transfection, Staining, Amplification, Binding Assay

Journal: Redox Biology

Article Title: Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells ☆

doi: 10.1016/j.redox.2017.03.002

Figure Lengend Snippet: eGFP in cells induces gene expression associated with oxidative stress and other biological processes. Images of HeLa cells and stable eGFP-expressing HeLa cells (HeLa/eGFP) under a fluorescent microscope with a green filter can be seen in (a). eGFP was also detected by Western blotting, with GAPDH staining as the loading control. In (b) extracellular H 2 O 2 was determined in HeLa and HeLa/eGFP. Catalase was added to cultures at 500 U/mL. (c) Microarray-based experiments were designed to show the effect of GFP expression in the gene expression of two independent cell models. The two GSEA (Gene Set Enrichment Analyses ) were performed using the hallmark dataset (h.all.v5.0.symbols) and 1000 permutations. The contrasts were HeLa/eGFP versus HeLa, and Hela.tet.eGFP + doxycycline subtracted from HeLa.tet.eGFP versus HeLa.tet + doxycycline subtracted from HeLa.tet. The enriched gene sets with p -values lower than 0.001 were ranked, and matches between the two independent analyses are shown highlighted in yellow. The top genes contributing to the enrichment for the commonly enriched gene sets are depicted as heat maps in . (d) Intracellular levels of HIF1α were determined by Western blotting using whole cell lysates of HeLa cells and HeLa cells stably expressing eGFP. In (e), GAPDH densitometry was used as the loading control to normalize the densitometry of HIF1α. * is used to show statistically significant differences between HeLa and HeLa/eGFP.

Article Snippet: HeLa.tet and HeLa.tet.eGFP were purchased from GenTarget Inc. (San Diego, CA) and were also certified to be free of mycoplasma.

Techniques: Expressing, Microscopy, Western Blot, Staining, Microarray, Stable Transfection

Journal:

Article Title: The kinase haspin is required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase chromosome alignment

doi: 10.1101/gad.1267105

Figure Lengend Snippet: Haspin is phosphorylated during mitosis, and its overexpression delays mitotic progression. (A) HeLa Tet-On/myc-haspin- or vector-alone-stably-transfected cells were induced with 1 μg/mL doxycycline for the times indicated, and myc-haspin expression was assessed by rabbit antihaspin immunoblotting. (B) HeLa Tet-On/myc-haspin- or vector-alone-stably-transfected cells were induced with 1 μg/mL doxycycline for 24 h before synchronization at G1/S and analysis as described for Figure 4B. (C) Synchronized HeLa Tet-On/myc-haspin cells as shown in B were lysed and analyzed by immunoblotting and haspin in vitro kinase assay with γ32P-ATP using H3–GST or H3-T3A–GST as a control. (D) Synchronized HeLa Tet-On/vector cells as shown in B were analyzed as described in C.(E) Induced HeLa Tet-On/myc-haspin cells were fractionated into looselyadherent mitotic (mitosis) and adherent interphase-enriched (interphase) populations or treated with colcemid prior to lysis. Rabbit antihaspin or rabbit IgG control immunoprecipitates were analyzed by rabbit antihaspin immunoblotting. (F) Induced HeLa Tet-On/myc-haspin cells were treated colcemid for 12 h prior to release. Whole-cell lysates made at the times indicated were analyzed by antihaspin immunoblotting. (G) Induced HeLa Tet-On/myc-haspin cells were treated colcemid for 12 h or untreated prior to cell lysis. Antihaspin immunoprecipitates were incubated with or without λ phosphatase as described in Materials and Methods and analyzed by antihaspin immunoblotting.

Article Snippet: Stable transfection of HeLa Tet-On (BD Clontech) cells was carried out using Lipofectin with Plus Reagent (Invitrogen).

Techniques: Over Expression, Plasmid Preparation, Stable Transfection, Transfection, Expressing, Western Blot, In Vitro, Kinase Assay, Lysis, Incubation

Journal:

Article Title: The kinase haspin is required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase chromosome alignment

doi: 10.1101/gad.1267105

Figure Lengend Snippet: Endogenous haspin is required for phosphorylation of histone H3 on Thr 3. (A) Immunoprecipitates with rabbit antihaspin or rabbit IgG negative control (neg) antibodies from HeLa cell lysates were subjected to in vitro kinase assays with γ32P-ATP and H3–GST or H3-T3A–GST as substrates. Coomassie blue staining was used to confirm equivalent levels of GST proteins. (B) Antihaspin and anti-aurora B immunoprecipitates from HeLa cell lysates were subjected to in vitro kinase reactions with γ32P-ATP and H3–GST, H3-T3A–GST, or H3-S10A–GST proteins as substrates. Coomassie blue staining was used to confirm equivalent levels of GST proteins. (C) The synchronized HeLa Tet-On/vector cells shown in Figure 5B were analyzed by haspin in vitro kinase assay described for Figure 5C. Note that this is a longer exposure of the autoradiogram shown in Figure 5D, carried out in order to visualize endogenous haspin activity. Similar results were obtained with HeLa cells (data not shown). (D) HeLa cells were transfected with haspin siRNA (Ambion ID #1093), control siRNA (Ambion 4611), or no siRNA. Approximately 30 h after transfection, the cells were incubated with nocodazole for 16 h (mitotic) or left untreated (asynchronous) prior to lysis. Antihaspin and anti-aurora B immunoprecipitates were subjected to in vitro kinase reactions with γ32P-ATP and H3–GST, H3-T3A–GST, or H3-S10A–GST as substrates, and lysates were immunoblotted with anti-phospho-H3 (Thr-3) or anti-phospho-H3 (Ser-10) antibodies. The anti-phospho-H3 (Thr-3) blot was stripped and reprobed with antihistone H3 antibodies. The increased haspin activity in nocodazole-treated cells was not a reproducible finding, as shown in B and C. (E) NIH3T3 cells were transfected with mouse haspin (Ambion ID 67120) or negative control (Ambion 4611) siRNAs and analyzed as described for D.

Article Snippet: Stable transfection of HeLa Tet-On (BD Clontech) cells was carried out using Lipofectin with Plus Reagent (Invitrogen).

Techniques: Negative Control, In Vitro, Staining, Plasmid Preparation, Kinase Assay, Activity Assay, Transfection, Incubation, Lysis