Journal: Antiviral research
Article Title: Distinct dual antiviral mechanism that enhances hepatitis B virus mutagenesis and reduces viral DNA synthesis
doi: 10.1016/j.antiviral.2019.104540
Figure Lengend Snippet: HepAD38 cells (1.0 X 105 cells per well) were plated in a 24-well plate 24 h prior to virus-producing induction. Five days following removal of tetracycline from cultured HepAD38 cells and cell treatment with 10 nM gemcitabine, 50 μM 5-aza-dC, 200 μM 5-aza-C or the combination of 5-aza-dC/5-aza-C with gemcitabine, HBV rcDNA was purified from cell culture supernatants. A DNA product of 714 bp (HBV sequence 87–800) was amplified by PCR. After gel purification, the 714 bp product was ligated to the pGEM-T vector. Ligated DNAs introduced into E. coli by transformation, and insert containing-vectors were subjected to DNA sequencing, with 70 −100 colonies were sequenced per each treatment (see Supplemental Table 1). Sequence alignments were done by using SeqMan assembly tool in the Lasergene 7 software package (DNAStar; Madison, WI). A. Mutation frequency analysis. Mutation frequency was calculated by the number of mutations divided by the total number of bases sequenced. The mutation frequency of the untreated was 1.46 × 10−4 mutations per basepair (see Supplemental Table 1). B. Mutation spectra analysis. The spectra of mutations from each experimental group is depected by a pie chart, which shows the main mutation types and percentages observed. The size of the pie chart was normalized to the mutation frequency of untreated controls. Results are shown from 3 independent experiments. The gray asterisks and brackets associating any two bars indicate the significant difference between the experimental pairs. The statistical significance compared to the untreated control is shown above the bar with black asterisks. Significance was analyzed with the “N-1” Chi-squared test; “ns” = not significant; “****” = p ≤ 0.0001; “**” = p ≤ 0.01; “*” = p ≤ 0.05. Abbreviation: gem = gemcitabine.
Article Snippet: Sequence alignments were performed using SeqMan assembler of Lasergene 7 software package (DNAStar; Madison, WI).
Techniques: Cell Culture, Purification, Sequencing, Amplification, Gel Purification, Plasmid Preparation, Transformation Assay, DNA Sequencing, Software, Mutagenesis
