LMP1-induced changes in expression levels of shelterin RNAs and proteins are reversible. (A) Quantitative reverse-transcriptase polymerase chain reaction analysis of the mRNA expression of indicated shelterin genes on LMP1 expression and suppression. The mRNA levels of indicated genes (in relative units) at days 1, 3, 7, and 14 are normalized with expression of 3 housekeeping genes to the LMP1-suppressed cells at each time point. Also shown are levels for the mRNAs at days 7 and 14 if the expression of LMP1 was suppressed at day 3 (burgundy bars) or at day 14 if LMP1 expression was suppressed at day 7 (blue bars). Bars show mean values ± standard deviation of 3 independent experiments. *P < .05; **P < .0001 by Student t test. (B) LMP1 induced changes in expression levels of indicated shelterin proteins. Upper panel: BJAB-tTA-LMP1 cells were induced for the expression of LMP1 for 3, 7, and 14 days by removal of tetracycline. Protein levels of LMP1, TRF2, POT1, and α-tubulin as loading control were analyzed by western blotting by using corresponding antibodies. Reduction of shelterin proteins is observed (TRF2 and POT1) when LMP1 expression is induced. The suppression of LMP1 at day 3 returns shelterin proteins to the initial level of expression at day 7 (red boxes), and the suppression of LMP1 induction at day 7 returns shelterin proteins to the initial level of expression at day 14. Representative results from 3 independent experiments are shown. (C) Combined telomere FISH (red) and TRF2 immunostaining (green). Panel Ci: BJAB-tTA-LMP1–suppressed transfectant at day 14 shows nuclear TRF2 either free (green) or associated with mainly midsized telomeres (red) resulting in an orange-yellow signal in mononuclear cells. Panel Cii: BJAB-tTA-LMP1–induced cell with RS-like morphology. Abundant small telomeres and aggregates but nearly absent TRF2 (green arrows) were observed. Nuclei were counterstained with DAPI (blue).