Fig. 4. Effect of decreased DDX6 abundance on replication of HCV RNA.

Huh7 cells were transfected with either control or DDX6-specific siRNAs. The following day untransfected and cells transfected with siRNAs were infected with JFH-1 at a moi of 0.01. Two days p.i. untransfected cells were incubated with 25 μM MK-0608 for 6 h. Untransfected cells and siRNA transfected cells were incubated with 700 μM 4-thiouridine for 2 h at 37 °C. TRIzol extracted RNA was incubated with MTSEA-biotin XX, and newly synthesized RNA separated from total RNA by streptavidin affinity purification. (A) Northern blot of total and newly synthesized HCV and cellular RNA. (B) Quantification of northern blot (Figure 4A) is from two independent experiments. Data were normalized to control siRNA. (C) Northern blot of total and newly synthesized HCV and cellular RNA of untreated orMK-0608 treated JFH-1 infected cells. (D) Quantification of northern blot (Figure 4C) is from three independent experiments. Data were normalized to the untreated cells. Data are represented as the fraction of newly synthesized RNA per amount of total RNA relative to the control siRNA. Error bars display the mean ± SD. Data were analyzed using an unpaired Student’s t-Test; n.s. not significant, *p < 0.05 and ***p < 0.001. 4-ThioU RNA, 4-thiouridine labeled RNA.