FIG. 8.

MKRN1 protects cells against WNV-induced cell death and inhibits WNV replication. (A) Effects of MKRN1 overexpression on WNV cytotoxicity. 293T cells transiently expressing MKRN1, MKRN1 H307E, or Hdm2 were seeded at 8 × 10⁵ in a 60-mm-diameter plate and infected with WNV at a multiplicity of infection of 1 PFU/cell. After 72 h, the cells were fixed, followed by PI staining. The stained cells were detected and analyzed via flow cytometry as described for Fig. 1C. (B) WNV replication in MKRN1-overexpressing 293T cells. MKRN1-, MKRN1 H307E-, or Hdm2-overexpressing 293T cells were seeded at 3.9 × 10⁵ cells in six-well tissue culture plates and then infected with WNV at a multiplicity of infection of 100 PFU/cell. Virus replication titers at 24, 36, 40, 44, and 64 h postinfection were determined using plaque assay on Vero cells. This experiment was repeated three times. WT, wild type. Error bars indicate standard deviations. (C) Ablation of MKRN1. 293T cells were transfected using MKRN1 siRNA. After 72 h, the levels of the endogenous MKRN1 were measured using anti-MKRN1 antibodies. (D) Effects of MKRN1 depletion against WNV cytotoxicity. 293T cells transfected with MKRN1 or control siRNA were seeded at 8 × 10⁵ in a 60-mm-diameter plate and infected with WNV at a multiplicity of infection of 1 PFU/cell. After 72 h, the cells were fixed, followed by PI staining. The stained cells were detected and analyzed by flow cytometry. (E) Effect of MKRN1 depletion on WNV replication. 293T cells transfected with control siRNA or MKRN1 siRNA were seeded at 3.9 × 10⁵ cells in six-well tissue culture plates and then infected with WNV at a multiplicity of infection of 100 PFU/cell. Virus replication titers at 24, 36, 40, 44, and 64 h postinfection were determined using plaque assay on Vero cells. This experiment was repeated three times. Error bars indicate standard deviations.