Suppression of HBV replication in hepatocytes by TLR response activation in macrophages. (A) Schematic representation of the assay strategy used. (B) AML12HBV10 cells were seeded into a 12-well plate at a density of 1 × 105/well and cultured in the absence of tetracycline. Twenty-four hours later, the cells were treated with the TLR agonist at the indicated concentrations (direct treatment). Alternatively, AML12HBV10 cells were treated with media containing 50% of the conditioned media harvested from TLR agonist-treated RAW264.7 cells (cultured in a 12-well plate at a density of 5 × 105/well and treated for 12 h with the TLR agonist at the indicated concentrations) (indirect treatment). Cytoplasmic HBV core DNA was analyzed by Southern blot hybridization 2 days posttreatment. RC, relaxed circular DNA; DSL, double-stranded linear DNA; SS, single-stranded DNA; NT, no-treatment control, which consisted of samples from mock-treated AML12HBV10 cells (direct-treatment control) or from AML12HBV10 cells treated with 50% of the media from mock-treated RAW264.7 cells (indirect-treatment control).