Journal: The Journal of Biological Chemistry

Article Title: Heat shock proteins stimulate APOBEC-3–mediated cytidine deamination in the hepatitis B virus

doi: 10.1074/jbc.M116.760637

Figure Lengend Snippet: Effect of Hsp90 or Hsp70 inhibitor on A3G and A3B activity. Hsp inhibitors were used to evaluate the Hsp90 or Hsp70 effect on A3G and A3B. APOBEC-3s with or without Hsp90β were co-transfected with HBV into HepG2 cells in the presence or absence of Hsp90 inhibitor 17-AAG or Hsp70 inhibitor PES. After 2 days of treatment, HBV DNA mutation rates were determined by primer extension with the 88 °C 3D-PCR for A3G or 94 °C PCR for A3B. A, effect of Hsp90 inhibitor 17-AAG on A3G + Hsp90β mutation activity by pe1664; B, effect of Hsp90 inhibitor 17-AAG on A3B mutation activity by pe1674; C, effect of Hsp70 inhibitor PES on A3B mutational activity by pe1674. The data are presented graphically on the right. Each bar represents the average of triplicates for each treatment. Vector, background control with mock vector co-transfection; asterisks, statistically significant differences comparing treatments with controls: **, 0.01 < p < 0.001; *, 0.05 < p < 0.01. The one-factor ANOVA statistical test was used.

Article Snippet: Hsp40 (DNAJB1), Hsp70 (HSPA1A), Hsp90α, Hsp90β, Hsp104, and Hsp60 genes were purchased from Origene or Addgene.

Techniques: Activity Assay, Transfection, Mutagenesis, Plasmid Preparation, Cotransfection

Journal: The Journal of Biological Chemistry

Article Title: Heat shock proteins stimulate APOBEC-3–mediated cytidine deamination in the hepatitis B virus

doi: 10.1074/jbc.M116.760637

Figure Lengend Snippet: Effect of Hsp knockdown through siRNA on HBV mutation frequency. Each Hsp siRNA was transfected into HepG2 cells by a reverse transfection method with Lipofectamine RNAiMax. After a 24-h siRNA treatment, the cells were transfected with an HBV viral genome-encoding plasmid with or without A3G. The HepG2 cells were harvested for RNA or HBV extraction 24 h after HBV transfection. A, Hsp mRNA levels after siRNA treatment by quantitative RT-PCR determination. The relative mRNA levels for each siRNA treatment were determined by quantitative RT-PCR, using GAPDH as an internal reference. The mRNA levels relative to control were calculated and are represented graphically as percentages with the control as 100%. Each bar represents the average of triplicates for each treatment. Blank, background control by a scrambled negative siRNA without A3G co-transfection. Control, another control by the scrambled negative siRNA with A3G co-transfection. B, Hsp protein expression level analyses after siRNA treatment. Total cellular proteins were extracted from HepG2 cells after siRNA treatment, and Hsp protein levels in the cell lysates were analyzed by Western blotting with antibodies against endogenous Hsp90β, Hsp90α, Hsp70, and Hsp40. GAPDH was analyzed as the protein loading reference. C, HBV DNA mutation analyses. HBV DNAs were extracted from the cell lysates after a 24-h HBV transfection, and the resultant HBV DNA mutations were determined by pe1453 using an 88 °C 3D-PCR. The data are presented graphically on the right. Each bar represents the average of triplicates for each treatment. Asterisks, statistically significant differences comparing treatments with their corresponding control: **, 0.01 < p < 0.001; *, 0.05 < p < 0.01. The one-factor ANOVA statistical test was used.

Article Snippet: Hsp40 (DNAJB1), Hsp70 (HSPA1A), Hsp90α, Hsp90β, Hsp104, and Hsp60 genes were purchased from Origene or Addgene.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Quantitative RT-PCR, Cotransfection, Expressing, Western Blot