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. 2012 Apr;86(7):3934–3943. doi: 10.1128/JVI.05747-11

Fig 2.

Fig 2

The LMP1-DNs LMP1-TES1S and LMP1-TES2S display an antiproliferative effect on latency II EBV-transformed T-cell lines (NC5). (A) Effects of LMP1-DNs on the viability of NC5 cells or LCLs. Wild-type NC5 T cells and those with inducible GFP or with dominant negative LMP1-TES1S or LMP1-TES2S were counted. Then 2 × 105 cells for each assay were seeded twice in a medium with or without 2 μg/ml doxycycline for 24 h. LCLs transiently transfected with GFP, LMP1-TES1S, or LMP1-TES2S, or treated with BAY 11-7082, were counted. Then 2 × 105 cells for each assay were seeded. For each assay, cells were counted twice using trypan blue to analyze viability. (B) Effects of LMP1-DNs on cell growth. NC5 T cells with inducible GFP or with the DN LMP1-TES1S or LMP1-TES2S were counted and seeded as described above with or without 2 μg/ml doxycycline for 3 days. For each assay, cells were counted every 24 h for growth analysis. Results are shown as means ± standard deviations for 3 experiments carried out in triplicate. We compared induced and noninduced conditions for each cell line, and statistical analysis indicated that the growth of the NC5 T-cell line with inducible GFP was the same under both conditions, whereas the growth retardation of cells expressing LMP1-TES1S or LMP1-TES2S after doxycycline induction was statistically significant. (C) Effects of LMP1-DNs on cell proliferation. Wild-type NC5 T cells and those with inducible GFP or dominant negative LMP1 with (open bars) or without (filled bars) doxycycline (2 μg/ml), and LCLs transiently transfected with GFP, LMP1-TES1S, or LMP1-TES2S, or treated with BAY 11-7082 (shaded bars), were counted. Then 5 × 104 cells for each assay were seeded twice in 96-well plates with 1 μCi/well of [methyl-3H]thymidine. The incorporation of thymidine was analyzed after 48 h. The results are shown as means ± standard deviations for triplicate experiments. (D) Cell death due to LMP1-DN induction is dose dependent. NC5 T cells with inducible LMP1-DNs or a control vector were incubated with the indicated dose of doxycycline (0, 0.5, or 2 μg/ml) for 24 h; then cell death was assessed by addition of 50 μg/ml propidium iodide (PI) to samples just before flow cytometric analysis. Percentages indicate the proportion of cells in each quadrant.