Endogenous Spindlin1 represses HBV transcription in the setting of infection.

<p>HepaRG cells stably expressing a shRNA control (shCtrl) or a pool of three shRNA directed against Spindlin1 (shSpin1) were established after transduction with the corresponding lentiviral vectors and selection with puromycin. Infections were then carried out with normalized amount of wild virus (HBV wt) or virus deficient for the expression of HBx (HBV X-). 8 days after infection, cells were harvested for total RNA extraction and cccDNA extraction. (<b>A</b>) HBV and pre-rRNA transcription were analyzed by RT-qPCR (left panel and middle panel respectively). Spindlin1 expression in shCtrl or shSpin1 HepaRG cells was analyzed by RT-qPCR (right panel). The relative transcription level was normalized with Roth2 cellular gene. Transcript level in shCtrl HepaRG cells infected with HBV wt was set to 1. Error bars represent SD of three independent experiments. (<b>B</b>) Nuclear DNA extraction. DNA was treated with plasmid safe before cccDNA amplification. The amount of cccDNA was quantified by qPCR and normalized to the amount of cyclin A. The cccDNA level in each condition was analyzed by qPCR using primers that amplify the cccDNA. The cccDNA level in shCtrl HepaRG cells infected with HBV wt was set at 1. Error bars represent SD of three independent experiments.