FIG 8.

MYC reduces NDRG1 phosphorylation by downregulating SGK1 expression. (A) MYC overexpression. Huh7.5.1 cells were transfected with either control or MYC overexpression vectors, and protein was isolated at 48 h posttransfection. Protein was detected by using quantitative Western blotting (Wes) with the indicated antibodies. (B) MYC overexpression further reduces NDRG1 levels in HCV-infected cells. Huh7.5.1 cells were transfected with control or MYC expression vectors as described above for panel A. At 24 h posttransfection, the cells were HCV infected for an additional 72 h. The cells and proteins were then prepared as described above for panel A. Uninfected control cells were harvested in parallel. (C) HCV infection decreases SGK1 mRNA expression. A HCV time course was performed as described in the legend of Fig. 2, and SGK1 mRNA levels were monitored by RT-qPCR. An uninfected control was performed in parallel. (D) HCV infection reduces SGK1 protein levels. A HCV time course was performed as described in the legend of Fig. 2. SGK1 and GSK3β levels were monitored with the indicated primary antibodies and secondary HRP conjugates. Uninfected controls were run in parallel. (E) HCV alters the promoter activity of NDRG1, MYC, and SGK1. Promoter constructs containing a luciferase reporter under the control of NDRG1, MYC, and SGK1 promoter regions were transfected into Huh7.5.1 cells. At 6 h posttransfection, the cells were infected with HCV. Luciferase activity was measured at 48 h postinfection. The GAPDH promoter construct was used as a control. (F) Knockdown of SGK1 increases HCV RNA levels. Knockdown was performed as described in the text, using a pool of siRNAs. Seventy-two hours later, cells were infected with HCV, total intracellular and extracellular RNAs were harvested at 48 h postinfection, and HCV RNA was detected by RT-qPCR. (G) Overexpression of MYC increases HCV RNA levels. Cells were transfected with control or MYC expression plasmids for 24 h and infected with HCV. Total intracellular and extracellular RNAs were harvested at 48 h postinfection, and HCV RNA was detected by RT-qPCR. Error bars indicate standard deviations. All experiments were performed three times, and the data from a representative experiment are shown. **, P ≤ 0.01; *, P ≤ 0.05 (compared to the negative controls).