Impact of ESCRT-I subunits TSG101 and VPS28 on HBV production.

<p><b>A.</b> HuH-7 cells were treated with control siRNA (siC) or two individual siRNAs targeting TSG101 or VPS28. Cells were subsequently transfected with the pHBV replicon. The knockdown efficacy of the siRNAs was analyzed by Western blotting of cellular lysates using TSG101- or VPS28-specific antibodies. Uniformity of sample loading was confirmed by probing the lysates with anti-β-actin antibodies. Relative protein expression values were determined by densitometric analysis and demonstrated in percent amount relative to control cells. To probe for cytotoxicity, cellular supernatants were assayed for LDH activity and indicated as factors of increase relative to control cells. <b>B.</b> The number of virions released into the cell culture supernatants were quantitated by real-time PCR of the viral genomes and demonstrated in percent amount relative to control siRNA-treated cells. Error bars indicate the standard deviations from the mean of four experiments measured in duplicates. <b>C.</b> Co-depletion effects of ESCRT-I-specific siRNAs. Control siRNA or the two siRNAs targeting either TSG101 or VPS28 were introduced into HuH-7 cells prior to transfection with pHBV. Cell lysates were prepared and subjected to Western blotting using anti-TSG101, anti-VPS28, and anti-ß-actin antibodies. TSG101- and VPS28-specific band intensities were quantitated and demonstrated in percent amount relative to siC-transfected cells.