Volume 44, Issue 4, Pages 663-670 (April 2006) Inhibition of multiple gene expression and virus replication of HBV by stable RNA interference in 2.2.15 cells  Xiangrong Ren, Guangbin Luo, Zhenhui Xie, Lingjun Zhou, Xiangping Kong, Anlong Xu  Journal of Hepatology  Volume 44, Issue 4, Pages 663-670 (April 2006) DOI: 10.1016/j.jhep.2005.10.029 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Integration of the shRNA-producing vectors in the 2.2.15-derived stably transfected cell lines. (A) Schematic illustrations of the pGK-siHBV (top) and control empty vector pGK-U6P (bottom). The locations of PCR primers are indicated. (B) PCR results (prime T7 and CPRM) showing the amplification of the 350bp fragment in all eight clones derived from transfection using pGK-siHBV (lane 1–8) but not the two clones derived from transfection using the control empty vector. M, marker. Journal of Hepatology 2006 44, 663-670DOI: (10.1016/j.jhep.2005.10.029) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Detection of HBsAg and HBeAg in the culture medium by ELISA. 2.2.15 cells were stably transfected with empty vector (Su6p-1 and Su6p-2) and the vector expressing shRNA against HBV (S11-1, S11-2, S11-3, S11-4, S11-5, S11-6, S11-8 and S11-9), respectively. HBsAg and HBeAg in the culture medium from these cells were assayed using ELISA method. The value of 2.2.15 cells was defined as 100. The amounts of stably transfected cells are presented as the percentage of the amount secreted by 2.2.15 cells. The data shown are mean±SD based on three independent experiments. Journal of Hepatology 2006 44, 663-670DOI: (10.1016/j.jhep.2005.10.029) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Immunocytochemical staining for HBsAg. 2.2.15 cells and stably transfected cells were seeded on cover slips in 12-well plate. Three days later, cells were fixed, immunostained for HBsAg and visuslized under microscope. Representative fields from each cell are shown (original magnification×200). (A) 2.2.15 cells. (B) 2.2.15 cells stably transfected with the empty vector (Su6p-2). (C) and (D) are 2.2.15 cells stably transfected with the vector expressing shRNA against HBV. [This figure appears in colour on the web.] Journal of Hepatology 2006 44, 663-670DOI: (10.1016/j.jhep.2005.10.029) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Immunocytochemical staining for HBcAg in stably transfected cells. 2.2.15 cells and stably transfected cells were seeded on cover slips in 12-well plate. Three days later, cells were fixed, immunostained for HBcAg and visuslized under microscope. Representative fields from each cell are shown (original magnification×200). (A) 2.2.15 cells. (B) 2.2.15 cells stably transfected with the empty vector (Su6p-2). (C) and (D) are 2.2.15 cells stably transfected with the vector expressing shRNA against HBV. [This figure appears in colour on the web.] Journal of Hepatology 2006 44, 663-670DOI: (10.1016/j.jhep.2005.10.029) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 Northern blot analysis on HBV mRNA in stably transfected cells. (A) Total RNA was extracted from 2.2.15 cells and 2.2.15 cells transfected with the empty vector (Su6p-2) or the vector expressing shRNA against HBV (S11-4, S11-8). After treated with DNase I, 20μg RNA was analyzed by Northern blot. The numbers on the left denote the positions of the major HBV transcripts. The 28s and 18s rRNAs were visualized under ultraviolet light for equal loading control. (B) The hybridization signals for the viral transcript were quantitatively evaluated by NIH image analysis software. Data are expressed as mean±SD relative to the value of 2.2.15 cells in three independent experiments. Journal of Hepatology 2006 44, 663-670DOI: (10.1016/j.jhep.2005.10.029) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 6 Inhibition of HBV replication. (A) Real-time PCR analysis of HBV DNA. Representative data (mean±SD) from at least three independent experiments are shown. (B) Southern blot analysis of HBV DNA. Lane 1, parental 2.2.15 cells; lane 2, a 2.2.15-derived cell stable line with control empty vector pGK-U6P; lane 3–4, two 2.2.15-derived cell stable lines with the pGK-siHBV vector (S11-4 and S11-8). Bands corresponding to the relaxed circular (RC) and single-stranded (SS) HBV DNA replicative forms are indicated. (C) The hybridization signals for the viral core-associated DNA were quantitatively evaluated by NIH image analysis software. Data are expressed as mean±SD relative to the value of 2.2.15 cells in three independent experiments. Journal of Hepatology 2006 44, 663-670DOI: (10.1016/j.jhep.2005.10.029) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions