Fig 6.

The unique internal LANA sequence is important for the retention of GFP expression from episomal TR-containing DNA. (A) Ten million BJAB cells alone or BJAB cells stably expressing LANA or the different LANA deletion mutants were transfected with 5 × 10¹⁰ copies (5,000 copies per cell) of p2TR-GFP. Eighteen to 20 h posttransfection, cells were sorted for GFP expression and were seeded at similar densities. GFP expression was monitored daily by FACS for 14 days. To account for some small differences in cell growth after sorting, GFP expression was compared at a time point when cells had reached a concentration e^1.5 times that at day 1 of seeding. P values for comparisons between the indicated groups are shown. (B) Cell lines were assessed as for panel A but were initially transfected with p2TR-ΔRE-GFP, which is identical to p2TR-GFP except for deletion of the RE, abolishing LANA-mediated DNA replication. GFP expression was monitored for 7 days. The results in panels A and B are each from six experiments. (C) Western blotting of BJAB cells alone or BJAB cells expressing LANA or LANA mutants at the time of DNA segregation. (Left) (Top) Anti-T7 epitope blot; (bottom) anti-tubulin blot. (Right) Anti-LANA blot. Brightness and contrast were uniformly adjusted within each panel with Adobe Photoshop. Approximately 3.5 × 10⁵ cells were loaded per lane for the anti-T7 and anti-tubulin blots, and ∼1.0 × 10⁵ cells were loaded per lane for the anti-LANA blot. (D) GFP NLS, GFP LANA 1-32, GFP LANA 1-331, GFP LANA 1-331 GMR, and GFP LANA 33-331 were each expressed in BJAB cells. Cells were arrested in metaphase with colcemid. GFP is green, and chromosomes were counterstained with propidium iodide (red). The overlay of green and red generates yellow. Brightness and contrast in individual panels were uniformly adjusted using Adobe Photoshop. Magnification, ×630.