Figure 1.
Immunofluorescence imaging of PML NBs in NPC cell lines.
Log phase cells were fixed and stained for EBNA1 (red) and PML (green). The number of PML foci seen per cell was counted for 100 cells for each sample in three separate experiments and the average number with standard deviation is shown in the histograms, where *** denotes p values less than 0.0001 relative to the parental cell line. Exposure times of image capture were constant for all samples with the same antibody treatment. (A) EBV-positive C666-1 cells before and after treatment with siRNA against GFP (siGFP) or EBNA1 (siEBNA1) are shown. Arrowheads indicate a siEBNA1 treated cell that continued to express EBNA1 and can be used for comparison to neighboring silenced cells. (B) EBV-negative CNE2 and HK1 cell lines with (CNE2E, HK1E) and without stable EBNA1 expression are shown. CNE2E are also shown after silencing of EBNA1 expression where one of the three cells shown continues to express EBNA1 (arrowhead).
Figure 2.
Transient expression of EBNA1 and EBNA1 mutants in CNE2 cells.
(A) CNE2 cells were transiently transfected with a plasmid expressing EBNA1 or EBNA1 mutants Δ325–376 or Δ395–450, then stained for EBNA1 and PML. Both EBNA1-expressing and nonexpressing cells are shown 48 hrs post transfection. Exposure times of image capture were constant for all samples with the same antibody treatment. (B) Numbers of PML NBs per cell were counted 48 hours after expression of wildtype EBNA1. Cells were categorized into low and high EBNA1 expression depending on the intensity of EBNA1 staining. ** indicates 0.0001<p<0.001 and *** indicates p<0.0001 relative to untransfected cells. (C) The number of PML NBs were counted for all cells in (A) expressing wildtype or mutant EBNA1 proteins.
Figure 3.
EBNA1 expression diminished PML protein levels.
(A) C666-1 cells were treated with siRNA against EBNA1 (siEBNA1) or GFP (siGFP) then equal amounts of whole cell lysates were Western blotted and probed with an antibody recognizing all PML isoforms, EBNA1 and actin. (B) Equal amounts of whole cell lysates from CNE2 and CNE2E cells were Western blotted and probed as in A. Lysates from CNE2E cells after one (+) or two (++) rounds of transfection with siRNA against EBNA1 are also shown (lanes 3 and 4). (C) Lysates from CNE2 cells 48 hrs after transfection with the indicated amounts of an EBNA1 expression plasmid (OriPE) or the empty plasmid (OriP) were Western blotted for PML, EBNA1, actin or Sp100. (D) CNE2E cells were treated with MG132 proteasomal inhibitor for 0, 8 or 10 hours then equal amounts of lysates were blotted for PML, EBNA1 and actin.
Figure 4.
Interaction of EBNA1 with PML.
(A) Immunofluorescence images of CNE2 cells transfected with pc3OriPE and of C666-1 cells are shown after staining for EBNA1 and PML. The transfected CNE2 cells shown are those expressing very low levels of EBNA1. (B) EBNA1 was immunoprecipitated from C666-1 cells with anti-EBNA1 antibody (IP:EBNA1). The starting lysate (Input) and protein remaining after IP (Post IP) are also shown, in each case representing 1/40th of the lysate used in IP. The same lysate was also treated with IgG beads as a negative control (IP:IgG). All samples were Western blotted using antibodies against EBNA1 or all PML isoforms. In the right panel, the positions of FLAG-tagged PML isoform I (FLAG-PML I) and PML isoform IV (FLAG-PML IV) expressed in C666-1 cells are shown by Western blotting with anti-FLAG antibody.
Figure 5.
Effect of USP7 silencing on PML degradation by EBNA1.
(A) CNE2E cells expressing EBNA1 were transfected with siRNA against USP7 or GFP (negative control) then stained for USP7 and PML. Exposure times of image capture were constant for all samples with the same antibody treatment. (B) Equal amounts of cell lysates from (A) were analysed by Western blotting with the indicated antibodies. siUSP7-1 and siUSP7-2 are duplicate samples treated with siRNA against USP7. (C) CNE2 cells were transfected with siRNA against GFP or USP7 (left panel) then were transfected with EBNA1 expression plasmid pc3OripE and stained for EBNA1 and PML (right panel). Exposure times of image capture were constant for all samples with the same antibody treatment. (D) Equal amounts of cell lysates from (C) were analysed by Western blotting after pretreatment with siGFP or siUSP7 followed by EBNA1 expression.
Figure 6.
EBNA1-induced disruption of PML NBs in Saos-2 cells.
p53-null Saos-2 cells were transiently transfected with the expression plasmid with or without the EBNA1 gene as in Figure 2. (A) Cells were stained for EBNA1, PML and DNA (DAPI) and visualized by fluorescence microscopy. The number of PML NBs per cell were counted and average numbers with standard deviations are shown in the histogram, where *** indicates p<0.0001. (B) Equal amounts of lysates from the transfected cells were analysed by Western blotting as in Figure 3.
Figure 7.
Effects of EBNA1 on p53 activation.
(A) CNE2 and CNE2E cells were treated with etoposide (+) or left untreated (−) and equal amounts of total cell lysates were analysed by SDS-PAGE and Western blotting for p53 acetylated on K382 and total p53. Actin loading controls are also shown. (B) Hela cells were transfected with a plasmid lacking (oriP) or expressing (oriPE) EBNA1 then were treated with etoposide (+) or left untreated (−). Equal amounts of cell lysate were then analysed by Western blotting as in A.
Figure 8.
Effects of EBNA1 on DNA repair, apoptosis and cell survival.
(A) CNE2 and CNE2E cells, before and after siRNA treatment for GFP (siGFP; negative control) or EBNA1 (siEBNA1), were treated with UV or etoposide or left untreated. 24 hrs later cells were fixed, stained with propidium idodide and analysed for DNA content by FACS. The percentage of cells in each cell cycle stage was determined using Modfit and is shown for each sample. (B) CNE2 and CNE2E cells were treated with etoposide then analysed by TUNEL assay. Average percentage of TUNEL-positive cells are shown from three experiments with standard deviation and 0.0001<p<0.001 (**). (C) CNE2 (grey) and CNE2E (black) cells were treated with etoposide (top graph) or UV (bottom graph) then grown for the indicated number of days. At each time point, cells were incubated with Trypan blue and the percentage of cells that excluded Trypan blue was determined. Experiments were performed in triplicate and average numbers with standard deviations are shown. The difference in cell survival with and without EBNA1 3 days post etoposide treatment is statistically significant with a p value of 0.05.
Figure 9.
Models of the EBNA1, USP7 and PML interactions.
Three possible interpretations of the data on EBNA1-PML and EBNA1-USP7 interactions at PML nuclear bodies are shown.