Fig. 4

Spontaneous lytic EBV reactivation in NPC43 during cell line establishment. a Changes of average EBV copy number in NPC43 cells during establishment and propagation determined by real-time PCR. Estimated EBV copy number per cell at PD 5 was about 100. Gradual decrease of EBV copy number was observed in NPC43 during propagation from PD 5 to 26. EBV copy number became relatively stable from PD 26 to 190 (around 10–20 per cell). Data are shown as mean ± SD from three independent experiments. b NPC43 at PD 10 was treated with different concentrations of Y-27632 and the EBV copy number per cell was estimated by real-time PCR. In the absence of Y-27632, the EBV copy number per cell increased to 417 which may be due to induction of lytic reactivation of EBV in infected NPC43 cells. The copy number was around 100 at the concentrations of 4 and 10 μM. When the concentration of Y-27632 was increased to 20 μM, the copy number was around 10–20 per cell; *p < 0.05 in a two-tailed t-test. Data are shown as mean ± SD from three independent experiments. c Expression of EBV lytic proteins in NPC43 (PD 22) cultured under different concentrations of Y-27632. Expression levels of lytic proteins (Rta, Zta, BALF5, EA-D and Gp350/220) was negatively correlated with the concentration of Y-27632. Expression of β-actin was the loading control. HONE1-EBV treated with SAHA was included in this western blotting as a positive control for induction of lytic EBV reactivation. d IF staining results revealing the expression of lytic EBV proteins. The expression of Zta, EA-D and BALF2 was examined in NPC43 cells at early passages cultured in different concentrations of Y-27632 (top panel). HONE1-EBV treated with TPA and NaBu was included in the IF staining as a positive control of lytic EBV reactivation (middle panel). Quantification of percentage of positive-stained cells was performed by cell number counting. Data are shown as mean ± SD from 10 different microscopic views with over 2000 cells included; *p < 0.05 in a two-tailed Z-test. Scale bar, 100 μm