HBV replication profiles in 1.3ES2 cells growing from proliferation to confluence. 1.3ES2 cells (2.5 × 106) were initially plated onto a 6 cm diameter Petri dish and cultured continuously for 24 days. The culture medium was refreshed every three days during the experimental period. (A) The cell number profiles were determined with the Trypan Blue exclusive method at the indicated time points. (B) Total RNA was extracted for Northern blot analysis and the expression profiles of HBV transcripts were determined. A GAPDH-specific probe was used to control for equal sample loading. (C) Cell lysates (100 μg) collected at the indicated time points were subjected to Western blot analysis for the detections of HBcAg and β-actin. (D) Equal amounts of cell lysates were subjected to native agarose gel electrophoresis for particle blot analysis. The encapsidated viral genomes were quantified by using an HBV-specific probe. (E) The intracellular cccDNA was collected by Hirt extraction at each time point. The cccDNA from equal amounts of cells was thermally denatured and then subjected to restriction enzyme digestion before electrophoresis and Southern blot analysis. The ssDNA represents the single-stranded viral DNA derived from the protein-free RC DNA by artificial heat denaturation. (F) The culture medium was collected at the indicated time points and the amounts of secreted HBeAg were determined by ELISA. (G) HBsAg production in the culture medium at each time point was analyzed by ELISA. (H) The secreted viruses were purified from the collected culture media, and the HBV titers were determined with quantitative RT-PCR analysis. (I) These growing cells were analyzed by FACS caliber at each time points and the cell cycle distributions were represented. Each time point represents the mean ± SD of triplicate experiments.