p53-p21^WAF1 pathway is not activated in p16-null LCL 3CHT cells following removal of 4HT and inactivation of EBNA3C.

<p>(<b>A</b>) Four p16-null LCL 3CHT lines were cultured for 21 days with (+) or without (−) 4HT. Protein extracts were separated by SDS-PAGE and western blotted for p53 or p21^WAF1. No consistent changes in expression of p53 or p21^WAF1 were observed. (<b>B</b>) qPCR of cDNAs corresponding to p21^WAF1 transcripts in the same four p16-null LCL 3CHT lines (A1, A2, C1, C2) and two p16-competent LCL 3CHT lines (A and C) cultured for 21 days with or without 4HT. The data are normalized to the expression of control RNA.</p

EBNA3A and EBNA3C regulate p57^KIP2 expression.

<p>(<b>A</b>) Western blot showing the level of p57^KIP2 expression in four EBNA3A-KO (KO) LCLs and their revertant equivalent (REV) (used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005031#ppat.1005031.g001" target="_blank">Fig 1</a>). Below each Western blot is shown the level of p57^KIP2 mRNA determined by qPCR, normalized to GN2BL1 and relative to each “wild type” cell LCL EBNA3A-REV (3A-REV) of which the mRNA level is set to 1. (<b>B</b>) As in (A) but using five EBNA3A-ERT2 conditional LCLs cultured with (+) or without (-) 4HT (used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005031#ppat.1005031.g004" target="_blank">Fig 4</a>). (<b>C</b>) Western blot showing the expression of p57^KIP2 protein (left panel) and qPCR showing the level of p57^KIP2 mRNA (right panel) in two p16-null LCL 3CHT cultured with (+) or without (-) 4HT (used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005031#ppat.1005031.g001" target="_blank">Fig 1</a>).</p

Cell proliferation and viability after primary infection of B cells.

<p>B cells isolated using anti-CD19 magnetic beads (<b>A and B</b>) or ficoll-purified bulk lymphocytes from a buffy-coat residue (from donor D11) (<b>C and D</b>) were infected by the viruses indicated, at day zero. The proportion of proliferating cells (<b>A and C</b>) was assessed by EdU incorporation over 16 hours. Cell viability was assessed by Live/Dead staining (<b>B and D</b>). The sampling time after infection, and the virus used to infect the cells are as indicated. Note that FACS statistics were not collected for 3CHT+HT at 27 days due to a technical failure during automated data acquisition (_*). However visual inspection indicated that the majority of the cells were viable and this was consistent with the yield of RNA recovered from these cells (which was comparable to the WT and revertant infections – see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003187#ppat-1003187-g007" target="_blank">figure 7</a>).</p

Quantity and phosphorylation state of the ‘pocket’ proteins and expression of E2F1-target genes in p16-null and -competent LCL 3CHT lines with or without 4HT.

<p>(<b>A</b>) Similar cell extracts to those used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003187#ppat-1003187-g001" target="_blank">Figure 1D</a> were separated by SDS-PAGE and western blotted using a pan-specific anti-Rb antibody (pan-Rb) to detect all forms of Rb from hypophosphorylated (lower bands) to hyperphosphorylated (upper bands). Anti-phospho-Rb antibody (Rb-PO₄) detects only the hyperphosphorylated forms of Rb and anti-p107 indicates the pocket protein p107. Gamma-tubulin was used as a loading control. In p16-competent LCL 3CHT lines cultured for 21 days without 4HT, Rb is down-regulated and hypophosphorylated and p107 is also down-regulated. In p16-null LCL 3CHT lines (A2 and C2 in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003187#ppat-1003187-g001" target="_blank">Figure 1</a>), no difference in the quantity of Rb or p107 nor Rb phosphorylation is detectable regardless of EBNA3C activity. (<b>B</b>) Regulation of the E2F1-target EZH2. Samples of protein extracts used in (A) were separated by SDS-PAGE and western blotted with an antibody specific for the polycomb complex subunit EZH2 – a protein induced by E2F1. Again gamma-tubulin was used as a loading control. All these data are representative of two independent experiments each including at least two p16-null LCL 3CHT lines. (<b>C</b>) Heat-map visualization of microarray data from p16^INK4a-null 3CHT LCLs grown in the presence or absence of 4HT for more than 30 days. This displays relative transcript levels (i.e. raw transcript levels corrected for variation between the three cell clones and then mean-normalized for each gene) for E2F1 and 45 of its transcriptional targets. The scale indicates high (red) to low (blue) changes in transcript levels, with the extremes of the scale indicating a 0.42-fold change in expression (ie a sample on the extreme red end of the scale would show a 1.42-fold expression increase over one at the center - grey). The only gene significantly and consistently altered in its expression by the inactivation of EBNA3C is TP73 (indicated by arrow), which falls upon EBNA3C inactivation. The dendrogram (bottom) shows the ordering of the genes by their clustering into co-regulated groups. Cell line clone IDs and treatment (±4HT) are indicated to the left.</p

EBNA3A and EBNA3C induce chromosome looping at the miR-221/miR-222 cluster locus.

<p>(<b>A</b>) Schematic of the miR-221/miR-222 cluster locus depicts the location of both miRs, the HindIII sites, the 28kb pri-miR-221/222 transcription start site and primers used for the chromosome conformation capture assay. (<b>B</b>) EBNA3A-KO and EBNA3A-Rev LCLs (D3) were used for chromosome conformation analysis. Interaction between the promoter (P) of the 28kb pri-miR-221/222 and EBNA3A/3C binding site 2 (BS2 –including BS2a and BS2b, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005031#ppat.1005031.g008" target="_blank">Fig 8A</a>) or 3 (BS3) is dependent on the presence of EBNA3A. PCR primers (NC) corresponding to a site located downstream of the miR-221/miR-222 locus were used as a negative control. Positive control (+ve control) showed PCR reactions using a DNA control template. (<b>C</b>) Same as (B) but using p16-null LCL 3CHT (LCL 3CHT never HT) cultured for 30 days with (LCL 3CHT +HT) or without 4HT. Interaction between P to BS2 and P to BS3 occurred only when EBNA3C is active (LCL 3CHT +HT). (<b>D</b>). Loading control primers L1 and L2 amplify DNA contained in a single HindIII fragment and have been used as DNA loading control between the DNA samples used for chromosome conformation capture. (<b>E</b>) Schematic model of chromatin loop formation induced by EBNA3A and EBNA3C at miR-221/miR-222 cluster locus.</p

Inactivation of miR-221 and miR-222 in LCLs with corresponding anti-miRs.

<p>LCLs were electroporated with the anti-miR indicated; p57^KIP2, p27^KIP1 p21^CIP1 and PUMA expression have been analysed by western blot. The blot was probed for γ-tubulin as an additional control for loading and a non-targeted protein. There were increases in 57^KIP2 and p27^KIP1, but not p21^CIP1 or PUMA in cells transfected with the specific LNA anti-miRs.</p

The increase of p57^KIP2 level is associated with de-phosphorylation of Rb and reduced entry into S phase and proliferation.

<p>(<b>A</b>) Western blot analysis of extracts from LCL EBNA3A-ERT2 (line 5) cultured with (+) or without (-) 4HT for 30 days showing that after inactivation (and degradation) of the EBNA3A-ERT2 fusion protein, p57^KIP2 expression increases, and the amount of hyperphosphorylated Rb (ppRb) is dramatically reduced and is no longer detected using a phospho-Rb-specific antibody. The blot was probed for γ-tubulin as a control for loading. (<b>B</b>) Cell cycle distribution of LCL EBNA3A-ERT2 (line 5) culture with or without 4HT for four weeks was determined by flow cytometry following exposure to EdU for 2 hours (2h pulse). (<b>C</b>) A comparison of the population growth rate between these cells cultured with or without 4HT was analysed by counting the number of viable cells every 2–3 days. Total cell numbers were plotted at each time point. Data are representative of two independent experiments.</p

Proliferation rate of p16-null and -competent LCL 3CHT lines following EBNA3C inactivation.

<p>(<b>A</b>) Representative FACS analysis of cells cultured for 22 days with or without 4HT, then incubated for 1 hour with BrdU and stained with anti-BrdU-FITC/PI. The gated population comprises the cells that have undergone DNA replication while incubated with BrdU (cells that have DNA content between 2N and 4N and highly incorporate BrdU). (<b>B</b>) BrdU incorporation in every cell line cultured without 4HT was normalized at each time-point to values measured in the respective cell line cultured with 4HT. Comparison of the proliferation of four p16-null LCL 3CHT lines (C1, C2, A1, A2; shown as diamonds) and two p16-competent LCL 3CHT lines [-C and -A, (line A in duplicate experiments); shown as squares], quantified by BrdU incorporation, in the time-courses of up to 30 days following removal of 4HT. The time interval between sample harvesting sometime differed between lines. The proliferation rate of the p16-competent LCL 3CHT lines cultured without 4HT progressively declines with time, while the proliferation of the p16-null LCL 3CHT lines without 4HT remains unaltered. Error bars indicate the standard deviations between experiments.</p

Activation of EBNA3C represses miR-143/miR-145 expression.

<p>(<b>A</b>) MiR-143, miR-145 and RNU48 expression were analysed by qPCR from p16-null LCL 3CHT established without the presence of 4HT (never HT) or 30 days after 4HT was added to culture medium (+HT). MiR levels in p16-null LCL 3CHT +HT are relative to the LCL never HT. (<b>B</b>) As in (A) but analysing the expression of ALAS1, ADAM28 and ADAMDEC1 by qPCR as controls for the activation of EBNA3C following the addition of 4HT.</p

Regulation of miRs in EBNA3A-conditional LCLs.

<p>(<b>A</b>) Five EBNA3A-ERT2 LCLs (established in the presence of 4HT and named from 1 to 5) were cultured for ~30 days with (+) or without (-) 4HT and EBNA3A-ERT2 protein expression was analysed by western blot. (<b>B</b>) MiR-221/miR-222 expression was determined by qPCR using total RNA extracted from the same five EBNA3A-ERT2 cell lines (LCLs 1, 2, 3, 4 and 5). (<b>C</b>) As in (B) but analysing miR-143 and miR-145 expression.</p