Figure 2

HBV infection of NTCP/Cas9 cells. (a) Expression of NTCP in HepG2 cells expressing Cas9. Cells were sorted by FACS to enrich for populations with high NTCP levels. Lane 1, cell pool prior to FACS; lane 2, control HepG2 cells; lanes 3 and 4, 5 and 70% fraction of FACS sorted cells with the highest GFP expression levels. (b) Infection of NTCP/Cas9 cells with HBV derived from HepAD38 cells. Cells were processed for IF analysis with HBcAg-specific antibody C1-5 8 days after infection. (c) Northern blot analysis of total RNA extracted from HepG2/NTCP cells infected with HBV (lane 3) and HepAD38 cells as a control (lane1). Uninfected cells (lane 2) and ribosomal 28S RNA served as a control for the amount of RNA loaded onto each well. Lane 4 is a longer exposure of lane 3. (d) Analysis of core DNA extracted from HepG2/NTCP (H-NT) cells infected with HBV. DNA was extracted 14 days after HBV infection (lanes 1 and 2). Cells were cultured without (lane 1) and with entecavir (ETV, 10 µg/ml)) (lane 2). DNA extracted from HepAD38 (AD38) cells served as a control (lanes 3 and 4). Lane 3 was loaded with 1/10th of the amount of DNA used for lane 2. (e) PCR analysis of HBV DNA extracted from infected NTCP/Cas9 cells. DNA was extracted with the Hirt procedure (H) from total cell extracts or from the cytoplasm (C) of the HBV-infected cells. Primers used for the PCR reactions are described in Supplementary Table S1. Ccc, CCC DNA–specific primers overlapping the cohesive overlap in rcDNA (Figure 1a); mito, mitochondrial DNA-specific primers; rc, primers for amplification of rcDNA. (f) Conditional expression of Cas9 in two different cell clones. Cells were cultured with (lanes 2 and 4) and without (lanes 1 and 3) dox. dox, doxycycline tub; tubulin.