Involvement of PUF60 in regulation of HBV RNA expression. (a) A schematic diagram of HBV RNAs and regions used as probes for northern blotting is indicated (top). pcDNA-F-PUF60 or an empty vector (EV) was co-transfected with pUC-HB-Ce into HuH-7 cells. At day 2 or 4 post-transfection (pt), total RNA was extracted from cells and separated on an agarose gel. HBV 3.5 kb RNA and spRNA (upper panels) and 3.5 kb RNA and HBs RNA (lower panels) were detected by northern blotting using probe PG (nt 1998–2447) and probe S (nt 3205–488), respectively. Band intensities of 3.5 kb RNA on the blots with PG probe were determined by Image-J software and those of control samples (EV) were calculated as 1. (b) Total RNAs prepared as described above were used for semi-quantitative RT-PCR with (RT(+)) or without (RT(−)) reverse transcription. cDNA bands corresponding to unspliced 3.5 kb RNA and its spliced forms (spRNAs) were detected by agarose gel electrophoresis. 18 S ribosomal RNA (18 S) was also detected. Immunoblotting indicated expression of PUF60 and GAPDH in transfected cells. (c) RT-qPCR analysis was performed to determine 3.5 kb RNA and spRNA levels in cells as described above. (d) Nuclear and cytoplasmic fractions of cells transfected with pUC-HB-Ce with pcDNA-F-PUF60 or EV were isolated and 3.5 kb RNA levels in each fraction were determined at days 1 and 4 pt. (e) Dose-dependent effect of PUF60 on 3.5 kb RNA levels was determined in cells transfected with pUC-HB-Ce with various concentrations of pcDNA-F-PUF60 by RT-qPCR. (f) Effect of PUF60 expression on 3.5 kb RNA levels of various HBV genotypes was determined in cells transfected with pcDNA-F-PUF60 and a plasmid carrying the 1.24-fold HBV genome derived from HBV genotype (GT) A, B or C. (c)–(f) Data are normalized to that of β-actin mRNA and values of “EV” (GT-A EV in case of (f)) are set to 1. Values shown represent means ± SD obtained from three independent samples. Statistical differences compared with the control (EV) are shown. **p < 0.01, Student’s t test. Full-length blots in (a) and (b) are presented in Supplementary Figures S14 and S15, respectively.