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. 2015 May 20;89(15):7905–7921. doi: 10.1128/JVI.00729-15

FIG 4.

FIG 4

Downregulation of p53 and FBP1 have opposite effects on p21 expression and HCV replication. (A) Upregulation of p21 in FBP1-kd Huh7.5 or HepG2 cells is abolished upon downregulation of p53. Control and FBP1-kd Huh7.5 cells (upper) or HepG2 cells (lower) were transfected with p53 siRNA and grown for 48 h. The normalized cell lysates were analyzed for the expression of FBP1, p53, p21, and actin by Western blotting. Lane 1, control cells; lane 2, vector control; lane 3, FBP1-kd cells; lanes 4 and 6, cells transfected with control siRNA; lanes 5 and 7, cells transfected with p53 siRNA. (B and C) Downregulation of p53 enhanced HCV replication in control and FBP1-kd Huh7.5 cells. Control Huh7.5 cells (B) and stable FBP1-kd Huh7.5 cells (C) were transfected with p53 siRNA and, 10 h later, infected with infectious HCV-JFH1 virions and then grown further for 72 h. Cell lysates were prepared, normalized for protein concentration, and Western blotted for the expression of NS5A, FBP1, p53, p21, and actin. Another set of cells was used for isolation of total RNA for quantitative real-time RT-PCR of JFH1 HCV RNA and GAPDH mRNA. (Left) Lane 1, control; lane 2, reagent control; lane 3, siRNA control; lane 4, p53 siRNA. (Right) Fold change in JFH1 HCV RNA concentration in control and FBP1-kd Huh7.5 cells quantified by real-time RT-PCR. (D) Transient (exp) expression of p53 suppresses HCV replication in p53-kd Huh7.5 and HepG2 cells. Stable p53-kd Huh7.5 cells (left) and HepG2 cells (right) were transfected with an shRNA-resistant expression clone of p53, and 10 h later, Huh7.5 cells were infected with HCV-JFH1 virions while HepG2 cells were transfected with MH14 HCV replicon RNA. Cells were grown for 72 h; total RNA was isolated. The quantitative real-time RT-PCR for HCV RNA and mRNA levels of p53, p21, and FBP1 were carried out with GAPDH mRNA as the internal control. The Western blot of p53 expression also is shown. Lanes 1 to 4, control cells; lane 5 to 8, p53-kd cells; lanes 9 to 12, p53-kd cells in which p53 was transiently expressed.