Journal: Cell reports

Article Title: KSHV episome tethering sites on host chromosomes and regulation of latency-lytic switch by CHD4

doi: 10.1016/j.celrep.2022.110788

Figure Lengend Snippet:

Article Snippet: Human: BC3 , ATCC , CRL-2277.

Techniques: Produced, Virus, Recombinant, Protease Inhibitor, Magnetic Beads, Luciferase, Sequencing, In Vitro, Software

Journal: Scientific Reports

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production

doi: 10.1038/s41598-017-00401-7

Figure Lengend Snippet: Construction of recombinant ΔORF34 KSHV BAC and its revertant. ( a ) Schematic illustration of the KSHV genome including the ORF34 coding region. Using a two-step Red recombination system, three stop codons were inserted into the ORF34 coding region of KSHV BAC16 (nt54653 – nt54654; Accession number: GQ994935) to construct ORF34-deficient BAC clone (ΔORF34-BAC16). The stop codons were replaced with the original wild-type sequence for revertant BAC clone (Revertant-BAC16). ( b ) Agarose-gel electrophoresis of the recombinant KSHV BACmids, digested with EcoRI. The asterisks (*) indicate Insertion and deletion of a kanamycin-resistance cassette in each BAC clone. Original data is shown in Supplementary Fig. . ( c ) DNA sequencing results of ORF34 mutagenesis sites in ΔORF34-BAC16 and Revertant-BAC16.

Article Snippet: To construct pENTR4-KSHV RTA/ORF50, KSHV RTA/ORF50 fragments were obtained by PCR from KSHV genome harboring PEL cells (BC3) and inserted into pENTR4 no ccdB (686-1), a gift from Dr. Eric Campeau (Addgene plasmid # 17424) .

Techniques: Recombinant, Construct, Sequencing, Agarose Gel Electrophoresis, DNA Sequencing, Mutagenesis

Journal: Scientific Reports

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production

doi: 10.1038/s41598-017-00401-7

Figure Lengend Snippet: ORF34 is essential for virus production but not DNA replication of KSHV. ( a ) The effects of ORF34 deficiency on recombinant KSHV production. Each iVero cell line (iVero-WT, iVero-ΔORF34, or iVero-Revertant) were treated with Dox and NaB to induce the lytic-cycle and viral production, and cultured media was harvested after 96 h. Viral precipitates were inoculated onto cells (Vero and 293T) and infection with each KSHV was carried out for 48 h. GFP positive cells were analyzed by flow-cytometer to determine the infectivity of the recombinant viruses. The forward scatter/side scatter plots and gates of Fig. 2a are shown in Supplementary Fig. . ( b ) Virus production in iVero-WT, iVero-ΔORF34, iVero-Revertant. Each iVero cell line was cultured for 48 h with medium containing of NaB and Dox. KSHV DNA were purified from capsidated KSHV virions in culture supernatants, and KSHV genome copies were determined by real-time PCR. ( c ) KSHV DNA replication in iVero-WT, iVero-ΔORF34, and iVero-Revertant cells. The iVero cells were cultured for 48 h with medium containing of NaB and Dox. Cellular genome containing KSHV genome DNA were purified from each cell lines. KSHV genome copies were determined by real-time PCR and normalized by the total DNA amount. ( d ) Rescue of virus production in iVero-ΔORF34 cells by exogenous ORF34 expression. The iVero-WT or iVero-ΔORF34 cells were transfected with control plasmid or ORF34 plasmids. After 2 days, transfected cells were cultured with NaB and Dox-containing medium for 3 days, and culture supernatant containing virus was harvested. The KSHV genome was quantified by real-time PCR. ( b – d ) Three or four independent samples were evaluated by real-time PCR. The error bars indicate standard deviations.

Article Snippet: To construct pENTR4-KSHV RTA/ORF50, KSHV RTA/ORF50 fragments were obtained by PCR from KSHV genome harboring PEL cells (BC3) and inserted into pENTR4 no ccdB (686-1), a gift from Dr. Eric Campeau (Addgene plasmid # 17424) .

Techniques: Virus, Recombinant, Cell Culture, Infection, Flow Cytometry, Purification, Real-time Polymerase Chain Reaction, Expressing, Transfection, Control, Plasmid Preparation

Journal: Scientific Reports

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production

doi: 10.1038/s41598-017-00401-7

Figure Lengend Snippet: ORF34 physically interacts with ORF24, ORF31, ORF18, ORF23, and ORF66. Cells (293T) were co-transfected with 2xS-tagged ORF34 and 6xMyc-ORF24 ( a ), 3xFLAG-ORF31 ( b ), 3xFLAG-ORF18 ( c ), 3xFLAG-ORF66 ( d ), 3xFLAG-ORF30 ( e ), or 6xMyc-ORF23 ( f ), upper panel) expression plasmids. Cell lysates were subjected to pull-down assays using S-protein-immobilized beads, which captures S-tagged ORF34. The precipitates were immunoblotted with anti-Myc or FLAG antibody to detect their interactions. ( e ) The pull-down sample was precipitated from cell extracts co-transfected with 2xS-ORF34 and 3xFLAG-ORF31, was applied in the left lane as the positive control. ( f ), lower panel) The pull-down sample was precipitated from cell extracts co-transfected with 2xS-ORF23 and 6xMyc-ORF34. ( a – f ) Original images of blotting data are shown in Supplementary Figs – . ( g ) Schematic depiction of the interactions between KSHV ORF34 and KSHV-PIC component candidates.

Article Snippet: To construct pENTR4-KSHV RTA/ORF50, KSHV RTA/ORF50 fragments were obtained by PCR from KSHV genome harboring PEL cells (BC3) and inserted into pENTR4 no ccdB (686-1), a gift from Dr. Eric Campeau (Addgene plasmid # 17424) .

Techniques: Transfection, Expressing, Positive Control

Journal: Scientific Reports

Article Title: Kaposi’s sarcoma-associated herpesvirus ORF34 is essential for late gene expression and virus production

doi: 10.1038/s41598-017-00401-7

Figure Lengend Snippet: The interaction of the ORF34 C-terminal region and ORF24 is essential for virus production. The iVero-ΔORF34 cells were transfected with control, ORF34 or a ORF34-deletion mutant plasmid. After 2 days, transfected cells were stimulated with NaB and Dox for 3 days. Progeny KSHV was purified from harvested culture supernatant, and the KSHV genome was quantified by real-time PCR. Viral productivities were assessed using three independent samples, and error bars indicate standard deviations. Statistical significance between each group and the control was analyzed by two-tailed Student’s t -test (N.S.: not significant, P > 0.05).

Article Snippet: To construct pENTR4-KSHV RTA/ORF50, KSHV RTA/ORF50 fragments were obtained by PCR from KSHV genome harboring PEL cells (BC3) and inserted into pENTR4 no ccdB (686-1), a gift from Dr. Eric Campeau (Addgene plasmid # 17424) .

Techniques: Virus, Transfection, Control, Mutagenesis, Plasmid Preparation, Purification, Real-time Polymerase Chain Reaction, Two Tailed Test

Journal:

Article Title: Activation of alternative NF-?B pathway by human herpes virus 8-encoded Fas-associated death domain-like IL-1?-converting enzyme inhibitory protein (vFLIP)

doi: 10.1073/pnas.0308016101

Figure Lengend Snippet: Status of alternating NF-κB pathway in vFLIP K13-expressing cells. (A) Alternative NF-κB pathway is constitutively active in PEL cell lines. Western blots demonstrating constitutive p100 up-regulation and processing in three PEL cell lines (BC-1, BC-3, and BCBL-1) as compared with CEM cells. The actin blot shows equal protein loading. The numbers below the blots represent the intensity of the p52 band relative to the actin band. (B) Expression of KSHV vFLIP K13, but not of other FLIPs, leads to p100 up-regulation and its processing into the p52 subunit. Cell lysates from H460 cells expressing an empty vector or the indicated Flag epitope-tagged FLIPs were analyzed by immunoblotting with an antibody against p52. The blot was reprobed with Flag and actin antibodies to demonstrate the expression of various FLIPs and equal loading of the lanes, respectively. (C and D) Retroviral-mediated expression of vFLIP K13 leads to p100 up-regulation and its processing into p52 subunit in HeLa and CEM cells, as measured by Western blotting by using a p52 antibody.

Article Snippet: PEL cell lines (BC-1, BC-3, and BCBL-1), CEM (T cell leukemia), BJAB (B-cell lymphoma), and HeLa (human ovarian carcinoma) were obtained from the American Type Culture Collection.

Techniques: Expressing, Western Blot, Plasmid Preparation, FLAG-tag, Retroviral

Journal:

Article Title: Activation of alternative NF-?B pathway by human herpes virus 8-encoded Fas-associated death domain-like IL-1?-converting enzyme inhibitory protein (vFLIP)

doi: 10.1073/pnas.0308016101

Figure Lengend Snippet: Effects of siRNA-mediated K13 down-regulation in a BC-3 PEL cell line. (A) Flow cytometry analysis demonstrating high-efficiency infection of BC-3 PEL cells with siRNA-encoding lentiviral vectors as determined by GFP expression. Analysis was performed 72 h after infection with the indicated lentiviral constructs. (B and C) BC-3 cells were left uninfected (UI) or infected with lentiviral vectors encoding a control siRNA or siRNAs directed against K13 and p100. Approximately 72 h after infection, cell lysates were prepared and used in Western blot with the indicated antibodies. Equal loading of protein was demonstrated by Western blotting with antibodies against actin or tubulin. The numbers below the blots represent the intensity of the K13 and p52 bands relative to tubulin and actin bands, respectively. (D and E) Effect of vFLIP-K13 and p100 gene silencing on BC-3 cell number. BC-3 cells were either left uninfected or infected with the indicated siRNA-encoding lentiviral constructs. At 72 h after infection, viable cell numbers were measured by using (3–4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (D), and Cell titer-Glo luminescence reagent (E). The data represents mean ± SD of a representative of three independent experiments with similar results. (F) Effect of vFLIP-K13 and p100 gene silencing on the proliferation of BC-3 cells. Cells were infected with the indicated siRNA-encoding lentiviral vectors and were grown for 24 h in the presence of BrdUrd reagent. ELISA was performed to detect the amount of BrdUrd-labeled DNA as suggested by the manufacturer (Exalpha Biologicals). The data represent mean ± SD of a representative of three independent experiments with similar results.

Article Snippet: PEL cell lines (BC-1, BC-3, and BCBL-1), CEM (T cell leukemia), BJAB (B-cell lymphoma), and HeLa (human ovarian carcinoma) were obtained from the American Type Culture Collection.

Techniques: Flow Cytometry, Infection, Expressing, Construct, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Labeling

Journal: Frontiers in Immunology

Article Title: The Affinity of Elongated Membrane-Tethered Ligands Determines Potency of T Cell Receptor Triggering

doi: 10.3389/fimmu.2017.00793

Figure Lengend Snippet: Intercellular distances between T cells and 3T3 antigen-presenting cells (APCs) depends on tether dimensions. (A) Binding of serial dilutions of OKT3 scFv, BC3 scFv, or 2C11 scFv to Jurkat (OKT3 and BC3) or 2B4 (2C11) T cells was determined by ELISA. Results show scFv binding relative to maximum binding of the individual scFv. Maximum binding was estimated for 2C11 scFv because saturation was not reached even at the highest concentration (250 µg/ml) of 2C11-BGP scFv examined. (B) Images of the interphase between Jurkat and 3T3 cells expressing the indicated membrane-anchored OKT3 scFv (150,000× original magnification. Scale bars, 100 nm). (C) Estimated mean intercellular distance between Jurkat and 3T3 cells expressing OKT3 scFv. Intercellular distances were measured at 11–20 equally spaced distances from 9 to 15 independent T cell-APC conjugates for each tether ( n = 15, 15, 10, 9, 10, and 10 for 3T3 cells expressing OKT3-1, OKT3-2, OKT3-3, OKT-CD43, OKT3-7, and OKT3-CD44, respectively). The box and whisker plot shows the mean intercellular distances, the 10–90 percentile (box) and the minimum and maximum values (whiskers). Significant differences of intercellular distances between 3T3 cells expressing OKT3 scFv with different tethers are indicated (*** p < 0.0001).

Article Snippet: BALB/c 3T3 cells, HT29 human colon cancer cells, BC3 hybridoma cells, HB65 hybridoma cells, and Jurkat human T cells were from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Membrane, Whisker Assay

Journal: Frontiers in Immunology

Article Title: The Affinity of Elongated Membrane-Tethered Ligands Determines Potency of T Cell Receptor Triggering

doi: 10.3389/fimmu.2017.00793

Figure Lengend Snippet: T cell activation by membrane-tethered BC3 scFv depends weakly on tether dimensions. (A) Surface expression of membrane-tethered BC3 scFv on 3T3 cells was measured by FACS. BC3-1 Lo cells express low levels of BC3 scFv via the BGP tether (one Ig-like domain). (B) Calcein-AM-labeled Jurkat T cells were incubated with monolayers of 3T3 cells expressing membrane-tethered BC3 scFv in the presence of serial dilutions of BC3 IgG (dark symbols) or 10 µg/ml negative control IgG (light symbols). Unbound T cells were washed away and cellular fluorescence of the remaining bound cells was measured ( n = 3). The y -axis shows the mean fluorescence of bound T cells, whereas the x -axis represents the concentration of soluble BC3 IgG added normalized for the level of each membrane-tethered BC3 scFv, as determined by mean immunofluorescence intensity of the 3T3 antigen-presenting cells (APCs) stained with anti-HA antibody. Bars, SD. (C) 5 × 10 4 Jurkat T cells were incubated with the indicated numbers of BC3 APCs or control phOx-1 APCs for 24 h before the concentration of IL-2 in the culture medium was determined ( n = 3). Bars, SD. (D) 10 7 Jurkat T cells were incubated with 10 6 of BC3 APCs or control phox-1 APCs for the indicated times before cells were lysed and CD3ζ was immunoprecipitated. Total CD3ζ (right panel) or phosphorylated CD3ζ (left panel) levels were determined by immunoblotting with anti-CD3ζ or anti-phosphotyrosine antibodies, respectively. 5 × 10 4 human T cells were incubated with BC3 APCs for 24 h before T cell proliferation (E) or IFN-γ in the culture medium (F) was determined ( n = 3). Bars, SD.

Article Snippet: BALB/c 3T3 cells, HT29 human colon cancer cells, BC3 hybridoma cells, HB65 hybridoma cells, and Jurkat human T cells were from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activation Assay, Membrane, Expressing, Labeling, Incubation, Negative Control, Fluorescence, Concentration Assay, Immunofluorescence, Staining, Control, Immunoprecipitation, Western Blot

Journal: Cells

Article Title: Interaction Between Human Skeletal and Mesenchymal Stem Cells Under Physioxia Enhances Cartilage Organoid Formation: A Phenotypic, Molecular, and Functional Characterization

doi: 10.3390/cells14181423

Figure Lengend Snippet: Extracellular matrix (ECM) protein production in cartilage organoids: ( A ) Immunohistochemical detection of aggrecan, type II collagen, proteoglycan-4, type I collagen, and type X collagen in SSC-derived organoids cultured without (–IM) or with (+IM) chondrogenic induction medium. Images acquired with an Olympus microscope at 10× magnification. ( B ) Immunohistochemical detection of the same ECM proteins in MSC-derived organoids under the same conditions. ( C ) Immunohistochemical detection of ECM proteins in organoids derived from SSC–MSC co-cultures, with or without chondrogenic induction medium. Images acquired with an Olympus microscope at 10× magnification. ( D – F ) Semi-quantitative scoring of ECM protein expression (aggrecan, type II collagen, proteoglycan-4, type I collagen, and type X collagen) in SSC-derived organoids ( D ), MSC-derived organoids ( E ), and SSC–MSC co-culture organoids ( F ), cultured with or without chondrogenic induction medium. ( G ) Comparative scoring of aggrecan, type II collagen, and proteoglycan-4 expression between cartilage organoids and the NHAC-kn chondrocyte line. ( H ) Comparative scoring of type I and type X collagen expression between cartilage organoids and the NHAC-kn chondrocyte line. The statistical difference is generated by Group A ( G , H ). Statistical significance was determined using the Kruskal–Wallis test followed by multiple comparisons; * p < 0.05; *** p < 0.001; **** p < 0.0001. ns: non-significant.

Article Snippet: For immunohistochemistry (IHC), the Autostainer Link 48 automated system was used with the following antibodies: aggrecan (Clone BC-3, Novus ® , Novus Biologicals, Centennial, CO, USA), collagen I alpha 1 (Clone COL-1, Novus ® , Novus Biologicals, Centennial, CO, USA), collagen II (Clone 5B2.5, Novus ® , Novus Biologicals, Centennial, CO, USA), collagen X alpha 1 (Clone SR3302, Novus ® , Novus Biologicals, Centennial, CO, USA), and PRG4 (NBP1-19048, Novus ® , Novus Biologicals, Centennial, CO, USA).

Techniques: Immunohistochemical staining, Derivative Assay, Cell Culture, Microscopy, Expressing, Co-Culture Assay, Generated