Silencing of SAMHD1 increases HBV replication in resting HepG2.2.15 cells. ( a ) SAMHD1 or actin protein levels were detected in cell lysates from SAMHD1 knockdown or control HepG2.2.15 cells by Western blotting. ( b ) Phosphorylation on residue T592 of SAMHD1 and cyclin B1 expression were detected by Western blotting of lysates from control shRNA HepG2.2.15 cells cultured for 3 days in complete versus serum-free medium. ( c-e ) HepG2.2.15 cells stably expressing two different SAMHD1 shRNAs or a control shRNA were cultured in serum-free medium for 3 days. ( c ) Luciferase activity (relative luciferase units, RLU) was detected in cells 24 hours post infection with a single-round HIV-1-based lentiviral vector that encoded luciferase. ( d ) Equal cell growth and viability between different shRNA cell lines was assessed using ATPLite. ( e ) The amount of HBV DNA in the supernatant was determined by qPCR and normalized to the control shRNA. In ( c , d ), the mean ± standard error mean (SEM) of three biological replicates of one representative experiment is depicted. In ( e ), the fold-changes to negative control were calculated for each individual experiment based on the median of three biological replicates, each measured in three technical replicates. The mean ± SEM of the fold-changes of four independent experiments is depicted. Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p