Skip to main content
. 2019 Apr 26;15(4):e1007742. doi: 10.1371/journal.ppat.1007742

Fig 2. siRNA screening of host cellular DNA polymerases required for intracellular cccDNA amplification.

Fig 2

HepAD38 cells were cultured in the presence of 2 mM PFA from day 2 to day 6 after tet removal. At day 4, the cells were re-seeded and transfected with 10 pmol siRNA targeting different cellular DNA polymerases by using RNAiMAX. (A) Total RNA was extracted at 48 h after transfection. mRNA of each indicated DNA polymerase was quantified by RT-qPCR and presented as relative amount in comparison with that in cells transfected with scramble siRNA (mean ± SD; n = 3). (B) From 48 h to 72 h after siRNA transfection (day 6 to day 7 after tet removal), cccDNA synthesis was resumed by removal of PFA from culture medium. Hirt DNA was extracted and HBV DNA was detected by Southern blot assay. The intensity of HBV cccDNA band was measured by ImageJ and presented as relative amount in comparison with that in cells transfected with scramble siRNA. Data represent 3 independent experiments (mean ± SD). Data were analyzed by two-tailed Student’s t-test (unpaired), ** P < 0.01.