Figure 3

Determination of the half-life of cccDNA in different growth stages. (A) and (B) show the cell growth curve and cccDNA expression profile in the proliferating 1.3ES2 cells. The growth confluent cells were reseeded to initiate their proliferation, and 3TC was added at the day after plating. (A) The cell numbers were determined with the Trypan Blue exclusive method at the indicated time points. (B) The cccDNA before and after 3TC treatment was collected by Hirt extraction at each time point. The cccDNA was thermally denatured at 85°C for 5 min, then subjected to Eco RI digestion before electrophoresis on a 1.2% agarose gel and analyzed by Southern blot analysis. (C) and (D) show the cell growth curve and cccDNA expression profile in the confluent cells. After continuous culture for 12 days, the confluent cells were treated directly with 3TC from day 12 to day 42. (C) The cells numbers were determined with the Trypan Blue exclusive method at the indicated time points. (D) The Hirt extracts for each time point were thermally denatured at 85°C for 5 min, then subjected to Eco RI digestion before electrophoresis on a 1.2% agarose gel and analyzed by Southern blot analysis. The line charts represent the relative quantitative intensities of the cccDNA from each experiment.