Journal: Virology

Article Title: Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency.

doi: 10.1016/j.virol.2006.03.047

Figure Lengend Snippet: Fig. 2. ICN activates RTA promoter in a dose-dependent manner. (A) Scheme to show the Notch expression constructs used for luciferase assay. Delta E is encoded by a cDNA consisting of codons 1–22 fused to codons 1673–2555 and is predicted to result in the synthesis of a mature polypeptide with an amino terminus lying 61 amino acids external to the transmembrane domain. Delta EA has an additional deletion of ankyrin repeats. ICN is encoded by a cDNA consisting of the first two codons of NOTCH1 fused to codon 1770, which lies 24 amino acids internal to the transmembrane domain (Aster et al., 1997). In the diagram, proteolytic cleavage by furin at site S1 produces two subunits, ECN and NTM, which remain non-covalently associated at the cell surface. Sites S2 and S3 identify the sites of proteolytic cleavage mediated by metalloproteases and presenilins induced upon activation by ligand. Cleavage at S3 yields activated form of Notch ICN (Schroeter et al., 1998). (B) Luciferase assay: 1 million of U2OS cells were transfected with 0.5-μg RTA promoter reporter plasmid along with increasing amount of full-length Notch, Delta E, Delta EA or ICN expression vector (0 ng, 10 ng, 20 ng, 50 ng, 100 ng, 200 ng) by lipofectamine 2000. Twenty four hours post-transfection, cells were harvested and lysed for reporter assay.

Article Snippet: A human osteosarcoma cell line U2OS was obtained from the American Type Culture Collection.

Techniques: Expressing, Construct, Luciferase, Activation Assay, Transfection, Plasmid Preparation, Reporter Assay

Journal: Virology

Article Title: Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency.

doi: 10.1016/j.virol.2006.03.047

Figure Lengend Snippet: Fig. 5. Transcriptional activity of ICN on RTA promoters. The reporter plasmid pRpluc contains a 3-kb sequence upstream of the translational initiation site of the RTA gene that drives the expression of firefly luciferase. A series of truncation promoters named as pRplucΔ2570, pRplucΔ1490 and pRplucΔ1327 were made for deletion of RBP–Jκ binding sites (bottom panel). A fixed amount (0.5 μg) of the reporter plasmids was transfected or co-transfected into U2OS cell with 50 ng of pflu-ICN. The promoter activity was expressed as the fold activation relative to the reporters alone control. The means and standard deviations from three independent transfections are shown.

Article Snippet: A human osteosarcoma cell line U2OS was obtained from the American Type Culture Collection.

Techniques: Activity Assay, Plasmid Preparation, Sequencing, Expressing, Luciferase, Binding Assay, Transfection, Activation Assay, Control

Journal: Virology

Article Title: Intracellular-activated Notch1 can reactivate Kaposi's sarcoma-associated herpesvirus from latency.

doi: 10.1016/j.virol.2006.03.047

Figure Lengend Snippet: Fig. 7. (A) LANA represses ICN transactivation on RTA promoter. 1 million of U2OS cells were transfected with 0.5 μg RTA promoter reporter plasmid and 50 ng ICN expression vector along with increasing amount of LANA expression vector (0 ng, 10 ng, 20 ng, 50 ng, 100 ng, 200 ng) by lipofectamine 2000. Twenty four hours post-transfection, cells were harvested and lysed for reporter assay. (B) For each transfection, 15 million BCBL1 cells were transfected with mock, pA3M empty vector, and ICN with or without LANA. Twenty four hours post-transfection, total RNAwas collected from the cells. A total of 5 μg of RNA was used with the Superscript First Strand Synthesis system to construct cDNA. Real-time PCR was performed using the DyNAmo SYBR Green qPCR kit with β-actin as the standard. The PCR data are expressed as the Ct values for RTA transcription. Each sample was tested in triplicate for the calculation of the mean and standard deviation. The relative transcript abundance (Log molecules) based on the Ct values for RTA. (C) Twenty million of Vero cells stably infected with Bac36 were mock-transfected or transfected with empty vector pA3M, ICN, or ICN plus LANA. Four days post-transfection, supernatant from each transfection was harvested and concentrated for infection of 293 cells. Twenty four hours post-infection, cells were harvested for FACS analysis to detect GFP expression marking the virus existence.

Article Snippet: A human osteosarcoma cell line U2OS was obtained from the American Type Culture Collection.

Techniques: Transfection, Plasmid Preparation, Expressing, Reporter Assay, Construct, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation, Stable Transfection, Infection, Virus