Effect of covalent linkage of ubiquitin. Covalent linkage of ubiquitin blocks LMP1-mediated cellular signaling pathways. (A) (B) Summary of the relative NF-κB (A) and STAT (B) activity induced by either pEGFP-N1, LMP1-GFP, Ub-LMP1-GFP Ub-Ala/Gly-LMP1-GFP, or Ub-Gly/Met-LMP1-GFP as determined by quantitation of luciferase activity produced from a cotransfected reporter plasmid. The data were normalized for transfection efficiency by measuring GFP⁺ cells and then expressed relative to the activity obtained with the B95.8 LMP1 gene (100%) without subtracting the basal activity in control pEGFP-N1-transfected cells. Results are the mean standard deviation of at least 4 separate experiments; ^* denotes that values obtained for Ub-LMP1-GFP are statistically significant (P < .05) when compared with LMP1-GFP. (C) Kinetics of cell survival in control and LMP1-GFP transfectants of HaCaT cells. The percentage of cell viability was determined by trypan blue staining every second day for up to 6 days of culture in medium containing 1% FCS. Cell number is expressed as cells × 10³/mL and is shown as the percentage of cell growth with counts at day 0 being considered 100%. (D) Clonogenic assay for cell growth. Cells (10³) from control, LMP1-GFP-, and Ub-LMP1-GFP-expressing 3T3 cells were plated in each well of 6-well plates. After 10 days of cell growth, the resulting colonies were stained with crystal violet. The results shown are from a representative duplicate experiment. Original magnification × 10.