Luminescence counter

All reporters were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, cells were prepared (2 × 10⁵/well) for Zp and Rp transfection. The Zp or Rp reporters were mixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in Opti-MEM (Invitrogen, Carlsbad, CA, USA) for 20 min and then added to the cells. After 3 h of transfection, kaempferol (0, 10, 20 and 50 μM) was pre-treated or not for 1 h, and then TPA (40 ng/mL) and SB (3 mM) were added to induce EBV reactivation. After a further 24 h incubation, the cells were collected and lysed in HEPES buffer (0.1M HEPES, pH 7.8, 1% Triton X-100, 1 mM CaCl₂, and 1 mM MgCl₂). Equal amounts of the lysates and luciferase assay reagent II (Promega, Madison, WI, USA) were co-incubated for 10 min. The luciferase activity was measured using a luminescence counter (Packard). Each sample was quantified for the amount of β-actin expression compared to that of the controls for variation of samples (data not shown). The standard deviation of each sample was calculated from three independent experiments in duplicate.

The construction of the Zp and Rp reporter plasmids has been described in previous reports [76 (link), 106 (link), 111 (link)]. Zp and Rp reporter plasmids were transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Briefly, NA or TW01 cells were seeded (2×10⁵/well) for Zp and Rp activation by TPA/SB. The Zp or Rp plasmid, mixed with Lipofectamine 2000 (Invitrogen) in Opti-MEM medium (Invitrogen), was incubated for 20 min, then added to the culture wells containing the cells. After 3 hr transfection, luteolin was added or not for pre-treatment for 1 hr, and then TPA (40 ng/ml) plus SB (3 mM) or SB (3 mM) alone were added to induce EBV into the lytic cycle. After induction for 24 hr, the cells were lysed in 50 μl HEPES buffer (0.1M HEPES, pH 7.8, 1% Triton X-100, 1 mM CaCl₂ and 1 mM MgCl₂) and 25 μl of the lysates were combined with 25 μl of Luciferase Assay Reagent II (Promega) for 10 min incubation. Finally, the luciferase activity was measured using a luminescence counter (Packard). Each lysate sample was quantified for the expression of β-actin to control for variation in the amount of sample (data not shown). The mean and standard deviation of each sample were calculated from three independent experiments in duplicate.