Impact of ESCRT-II subunits EAP20, EAP30, and EAP45 on HBV production.

A. HuH-7 cells were treated with control siRNA (siC), a siRNA pool targeting EAP20, or two different siRNA duplexes directed against EAP30 or EAP45. After 48 h, cells were retransfected with pHBV, and lysates and supernatants were harvested 72 h later. To probe for the efficiency of the knockdowns, lysates were immunoblotted with antibodies against EAP20, EAP30, and EAP45. Identical sample loading was assessed by anti-β-actin Western blotting, and relative protein expression values were determined by densitometric analysis and demonstrated in percent amount relative to control cells. To probe for cell lysis, supernatants were assayed for LDH activity. B. Virions released into the cellular supernatants were quantified by real-time PCR of the HBV genomes. Error bars indicate the standard deviations from the mean of four experiments measured in duplicates. C. Kinetic and co-depletion effects of the ESCRT-II-specific siRNAs. Cells were treated with the indicated siRNAs for 48 h, transfected with pHBV and lysed after 24, 48, or 72 h DNA transfection. RNAi effects on the expression of EAP20, EAP30, and EAP45 were analyzed by specific immunoblotting. A non-specific band stained by the antisera served as a control for identical gel loading (LC).