Journal: PLoS ONE

Article Title: DNA Damage Signaling Is Induced in the Absence of Epstein—Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA

doi: 10.1371/journal.pone.0126088

Figure Lengend Snippet: 2089 cells were transfected with an empty vector (CMV) or a plasmid bearing the wild-type ZEBRA gene. Cells were fixed and double-stained for (A) ZEBRA and pATM (S1981), or (B) EA-D and p53BP1 (S1778). EA-D-positive cells with EA-D in a diffuse (B: ii) or globular pattern (B: iii) were identified. Scale bar = 10 μm. (C) The percentages of cells containing DNA damage markers or lytic EBV markers were determined and the results represented as means; n = 3. The percentages of total cells that were positive for pATM or p53BP1 were determined and the results represented as average percentages. * denotes P<0.05; P = 0.047 for pATM+ cells in ZEBRA vs CMV and P = 0.048 for p53BP1+ cells in ZEBRA vs CMV (C: i) .The percentages of total cells that were positive for ZEBRA, Rta, EA-D were determined and the results represented as average percentages (C: ii) . The percentages of pATM-positive cells that were also positive for ZEBRA or Rta, and the percentages of p53BP1-positive cells that were also positive for EA-D was determined and the results represented as average percentages (C: iii) . The percentages of ZEBRA-positive, Rta-positive, or EA-D-positive cells that were also positive for pATM or p53BP1 were determined and the results represented as average percentages (C: iv) . The differences between percentages in C: ii, C: iii, and C: iv were not statistically significant (P>0.05).

Article Snippet: EA-D was detected using the mouse monoclonal antibody R3.1 [ ].Rta and BFRF3 proteins were detected using rabbit polyclonal antisera described previously [ , ]. pATM (S1981) was detected with a mouse monoclonal antibody (Rockland Immunochemicals #200-301-400). γH2AX (S139) was detected with a mouse monoclonal antibody (Millipore #05–636).

Techniques: Transfection, Plasmid Preparation, Staining

Journal: PLoS ONE

Article Title: DNA Damage Signaling Is Induced in the Absence of Epstein—Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA

doi: 10.1371/journal.pone.0126088

Figure Lengend Snippet: (A) ΔG4ΔG5 cells were transfected with an empty vector (CMV); (A: i) , or a plasmid bearing the wild-type ZEBRA gene (A: ii) , or a plasmid bearing the wild-type BGLF4 gene (A: iii) , together with a plasmid bearing a membrane targeted GFP gene (mGFP) then fixed and stained for pATM (S1981). Scale bar = 10 μm. (B) The percentages of transfected cells (based on mGFP staining) that also contained pATM foci were determined and the results represented as averages; n = 2. * denotes P<0.05; P = 0.016 for CMV+mGFP versus ZEBRA+mGFP and P = 0.019 for CMV+mGFP versus BGLF4+mGFP. (C) Cell lysates were analyzed by immunoblots with antibodies against ZEBRA. A non-specific band (NS) is shown as a loading control.

Article Snippet: EA-D was detected using the mouse monoclonal antibody R3.1 [ ].Rta and BFRF3 proteins were detected using rabbit polyclonal antisera described previously [ , ]. pATM (S1981) was detected with a mouse monoclonal antibody (Rockland Immunochemicals #200-301-400). γH2AX (S139) was detected with a mouse monoclonal antibody (Millipore #05–636).

Techniques: Transfection, Plasmid Preparation, Membrane, Staining, Western Blot, Control

Journal: PLoS ONE

Article Title: DNA Damage Signaling Is Induced in the Absence of Epstein—Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA

doi: 10.1371/journal.pone.0126088

Figure Lengend Snippet: (A) Raji cells treated with TPA (A: i) or left untreated (UN) (A: ii) and HH514-16 cells treated with AZA (A: iii) , or with AZA and PAA (A: iv) , or left untreated (UN) (A: v) were double-stained for ZEBRA and pATM (S1981). (B) The percentages of total cells that were positive for both ZEBRA and pATM were obtained and the results represented as averages (n = 3). * denotes P<0.05 and ** denotes P<0.01; for the average percentages of ZEBRA+ cells P = 0.027 in TPA versus UN RAJI samples and P = 0.025 for AZA versus UN HH514-16 samples. For the average percentages of ZEBRA+ and pATM+ cells P = 0.021in TPA versus UN RAJI samples, P = 0.0070 in AZA versus UN, and P = 0.047 in AZA+PAA versus UN HH514-16 samples. Scale bar = 10 μm.

Article Snippet: EA-D was detected using the mouse monoclonal antibody R3.1 [ ].Rta and BFRF3 proteins were detected using rabbit polyclonal antisera described previously [ , ]. pATM (S1981) was detected with a mouse monoclonal antibody (Rockland Immunochemicals #200-301-400). γH2AX (S139) was detected with a mouse monoclonal antibody (Millipore #05–636).

Techniques: Staining

Journal: PLoS ONE

Article Title: DNA Damage Signaling Is Induced in the Absence of Epstein—Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA

doi: 10.1371/journal.pone.0126088

Figure Lengend Snippet: (A) 293 cells were transfected with a plasmid expression vector for ZEBRA. Cells were double-stained for ZEBRA and pATM (S1981). White arrows indicate cells expressing ZEBRA. The blue arrow indicates a cell positive for pATM. Scale bar = 10 μm. (B) 293 cells were co-transfected with a plasmid expressing a membrane-targeted EGFP-farnesylated construct (mGFP) and an empty vector (CMV) or an expression vector for ZEBRA and stained for pATM (S1981). The percentage of total cells that were positive for both pATM and mGFP was determined in blinded experiments and the results represented as average percentages (n = 4), ** denotes P<0.005; P = 0.0048 for ZEBRA versus CMV. (C) 293 cells were transfected with an empty vector (CMV) or a plasmid expression vector for ZEBRA. Cell lysates were analyzed by immunoblots with antibodies against γH2AX and ZEBRA and β-Actin (C: i) , or H2AX and β-Actin (C: ii). γH2AX fold stimulation (FS) levels are shown; n = 3, ** denotes P<0.005; P = 0.0014 for γH2AX FS in CMV versus ZEBRA 293 samples (C: iii) .

Article Snippet: EA-D was detected using the mouse monoclonal antibody R3.1 [ ].Rta and BFRF3 proteins were detected using rabbit polyclonal antisera described previously [ , ]. pATM (S1981) was detected with a mouse monoclonal antibody (Rockland Immunochemicals #200-301-400). γH2AX (S139) was detected with a mouse monoclonal antibody (Millipore #05–636).

Techniques: Transfection, Plasmid Preparation, Expressing, Staining, Membrane, Construct, Western Blot

Journal: PLoS ONE

Article Title: DNA Damage Signaling Is Induced in the Absence of Epstein—Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA

doi: 10.1371/journal.pone.0126088

Figure Lengend Snippet: (A) 293 cells co-transfected with an empty vector (CMV) and a membrane-targeted EGFP-farnesylated construct (mGFP) (A: i) or singly transfected with expression vectors for ZEBRA (A: ii) , Z(S186A) (A: iii) , Z(N182E) (A: iv) , Z(S186E) (A: v) , or Z(R183E) (A: vi) were fixed and double-stained for ZEBRA and pATM (S1981); Arrows indicate transfected cells expressing transfected genes or pATM or transfected genes and pATM. Detector settings were kept constant during confocal image acquisition. (B) 293 cells were fixed and stained for pATM (S1981) after co-transfection with a membrane-targeted EGFP-farnesylated construct (mGFP) and an empty vector (CMV), or expression vectors for ZEBRA, or Z(S186A), or Z(N182E), or Z(S186E), or Z(R183E). The percentage of total cells that were positive for both pATM and mGFP was determined in blinded experiments (n = 4). **P<0.01; P = 0.0001 for WTZ vs. CMV, 0.00017 for Z(S186A) vs. CMV, and 0.0013 for Z(N182E) versus CMV.

Article Snippet: EA-D was detected using the mouse monoclonal antibody R3.1 [ ].Rta and BFRF3 proteins were detected using rabbit polyclonal antisera described previously [ , ]. pATM (S1981) was detected with a mouse monoclonal antibody (Rockland Immunochemicals #200-301-400). γH2AX (S139) was detected with a mouse monoclonal antibody (Millipore #05–636).

Techniques: Transfection, Plasmid Preparation, Membrane, Construct, Expressing, Staining, Cotransfection