FIG 7.

Activation of HOXB9 gene expression in cells harboring full-genomic replicon RNA but not subgenomic replicon RNA. (A) Huh7OK1 cells were transfected with full- or subgenomic replicon RNA. Cells harboring full-genomic replicon RNA (F) or subgenomic replicon RNA (S) were established in medium containing 400 μg/ml G418. Total RNA was prepared from mock-transfected cells and cells harboring each replicon. HOXB9 mRNA, intracellular HCV RNA and GAPDH mRNA levels were evaluated by RT-sqPCR. NTC, no-template control. (B) Cell lysates were prepared from mock-transfected cells or individual replicon cells and were then subjected to Western blotting. (C) NS5A and the core protein in the mock-transfected or full-genomic and subgenomic replicon cells were stained with a mouse anti-core antibody or anti-NS5A antibody and were then stained with an AF488-conjugated goat anti-mouse IgG antibody. The stained samples were observed under fluorescence microscopy. (D) Cells from which the full-genomic replicon RNA (FC) or subgenomic replicon RNA (SC) was removed by treatment with daclatasvir were also stained with the anti-NS5A antibody and were then stained with DAPI. (E) Total RNA was prepared from M, F, S, FC, and SC cells. HCV RNA, HOXB9 mRNA, and GAPDH mRNA levels were evaluated by RT-sqPCR. The data shown in this figure are representative of three independent experiments. (F) Total RNA was prepared from mock-transfected cells (Mock) and cells harboring full-genomic (F) or subgenomic (S) replicon RNA. The mRNA levels of HOXA1, HOXA3, HOXB1, HOXB8, HOXC13, HOXD13, and GAPDH were evaluated by RT-sqPCR. NTC, no-template control. A1, A3, B1, B8, B9, C13, and D13 indicate HOXA1, HOXA3, HOXB1, HOXB8, HOXB9, HOXC13, and HOXD13.