HBV replication assay in HepAD38 cells.

HepAD38 cells were trypsinized and then washed three times with phosphate-buffered saline (PBS) to remove traces of Tet. Cells were seeded into 96-well plates in assay medium (DMEM–F-12 medium with 2% Tet-free FBS) and treated with compounds. Supernatants and cells were harvested at 5 days postinduction. Viral loads in supernatants and cells were quantified by TaqMan qPCR using a primer pair (5′-CTGTGCCTTGGGTGGCTTT-3′ and 5′-AAGGAAAGAAGTCAGAAGGCAAAA-3′) and a probe (5′-5HEX-CTCCACAGT-ZEN-AGCTCCAAATTCTTTATAAGGGTC-3IABkPQ-3′) specific for the core gene sequence.