Journal: Scientific Reports
Article Title: Restrictive influence of SAMHD1 on Hepatitis B Virus life cycle
Figure Lengend Snippet: SAMHD1 restricts HBV replication in infected HepG2-NTCP cells. Detection of ( a ) SAMHD1 or ( b ) NTCP in SAMHD1 knockdown or control HepG2-NTCP cells by Western blotting. Actin or GAPDH were used as loading controls. ( c ) Equal cell growth and viability was assessed was assessed using ATPLite. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( d ) Control shRNA cells were cultured for 10 days in complete medium supplemented (+) or not (−) with 2.5% DMSO. When indicated cells were infected with HBV 48 hours after start of DMSO treatment. Phosphorylation at residue T592 in SAMHD1, as well as expression of total SAMHD1, cyclin B1 and actin, were detected by Western blotting. ( e ) Cells were cultured for 10 days in 2.5% DMSO-containing medium and infected with a single-round luciferase HIV-1 virus. Luciferase activity was detected 24 hours post infection. Depicted are mean values (±SEM) of three biological replicates of one representative experiment out of three. ( f-i ) Cells were infected with HBV inoculum, and where indicated, the entry inhibitor MyrcludexB (MyrB, 200 nM), or the reverse transcription inhibitor lamivudine (Lami, 0,5 μM) were added. Cells were cultured in medium containing 2.5% DMSO. Ten days post infection, HBV DNA from the supernatant ( f ), intracellular total HBV DNA ( g ) and cccDNA ( h ) were quantified by qPCR. ( i ) The intracellular HBV RNAs levels were determined 10 days post infection by RT-qPCR using primer sets that amplify the indicated HBV RNAs (3.5 kb; 3.5 kb and 2.4 kb; 3.5 kb, 2.4 kb and 2.1 kb, or all four HBV RNAs). ( f ) For each individual experiment, fold-changes to the untreated control shRNA were calculated based on the median of three biological replicates, each measured in three technical replicates. The mean ± SEM of the fold-changes of three independent experiments is depicted. ( g – i ) For each independent experiment, fold-changes to untreated control shRNA were calculated based on the median of three technical replicates. The mean ± SEM of the fold-changes of at least 3 independent experiments is represented. ( f – i ) Statistical significance was determined using a one-way ANOVA with multiple comparisons according to Dunnett (*p
Article Snippet: For HBV total DNA, qPCR reactions were performed on undigested samples in SYBR green supermix (Bio-rad 172-5121) using HBV specific primers HBV-total-DNA-F and HBV-total-DNA-R (95 °C for 10 minutes, followed up by 95 °C for 10 seconds and 60 °C for 30 seconds for 40 cycles).
Techniques: Infection, Western Blot, shRNA, Cell Culture, Expressing, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR