Up-regulation of HBV core promoter activity induced by PUF60. (a) Effect of PUF60 expression on HBV or cellular promoter activities was analyzed by transfection of HuH-7 cells with the luciferase reporter carrying the entire core promoter (nt 900–1817), ENII/BCP (nt 1627–1817), preS1 promoter (nt 2707–2847), preS2/S promoter (nt 2937–3204), human ubiquitin C promoter or human elongation factor 1α promoter and pcDNA-F-PUF60 or empty vector (EV). Reporter activities in the cells were measured at 24 h pt. Values are normalized to total protein concentrations in cell lysates. (b) Knockdown effect of PUF60 on core promoter activity (left) and 3.5 kb RNA expression (middle) as well as knockdown efficiency of PUF60 (right) were assessed. At 2 days after introducing PUF60 siRNAs (siPUF60) or its negative control (siNC), HuH-7 cells were transfected with pGLHBp900/1817 or pUC-HB-Ce and then reporter activities and RNA levels, respectively, were measured after 2 days of further culture. PUF60 mRNA expression was also determined. (c) Effect of PUF60 deletion on activation of the core promoter was assessed. HuH-7 cells were transfected with pGLHBp900/1817 and a plasmid expressing either wild-type PUF60, PUF60-D1, -D2, -D3 or EV. Reporter activities were measured at day 1 pt. (a)–(c) Data are normalized to that of β-actin mRNA and the values in cells transfected with EV or siNC are set to 1. All assays were performed in triplicate and results are presented as means ± SD. Statistical differences compared with the control (EV or siNC) are shown. *p < 0.05, **p < 0.01, Student’s t test.