FIG. 6.

(A) siRNA knockdown of PML nuclear body-associated proteins. The siRNAs of Sp100, NDP52, or PML were transfected into Huh7 cells to knock down the endogenous protein expression. Two days later, siRNA transfection was repeated to enhance the extent of knockdown. The proteins were identified by immunoblotting (IB) using individual antibodies on different days after siRNA transfection. (B and C) Effects of NDP52, Sp100, and PML siRNA knockdown on HDV RNA synthesis (C) and HDAg production (B). The siRNA of PML, NDP52, or Sp100 or nonspecific (NS) siRNA was transfected into HuH7 cells. Two days after siRNA transfection, cells were transfected with HDV RNA and mRNA, followed by retransfection with the same siRNA 24 h later to maintain the protein knockdown. The relative levels of HDAg (B) and HDV RNA (C) in each reaction were determined at 48 h after the second siRNA transfection (see Materials and Methods). The proteins were detected by immunoblotting using specific antibodies (B). Actin was used as a protein sample loading control. The HDV RNA quantitative analysis (C) was performed by specific reverse transcription and real-time PCR as described in Materials and Methods. Error bars indicate standard deviations.