Fig. 3.

6-AN inhibited HBV antigen secretion and blocked virus transcription and replication. HepAD38 cells were treated with 0 μM, 12.5 μM, 25 μM 6-AN and 25 nM ETV, after 3, 6 and 9 days of treatment, the level of HBsAg secreted in medium, HBV core DNA in supernatant and cells, total RNAs were extracted (n = 5 per group). (a-b) The relative HBsAg secretion levels were examined by HBsAg ELISA and Dot blot assay. (c) HepAD38 cells were treated with 250 μM 6-AN for 6 days. Culture media were collected and tested for the secretion of human albumin and apolipoprotein B with ELISA. Medium from cells treated with entecavir 25 nM each were included as controls. (d-e) 6-AN inhibited HBV transcription dose-dependently in HepAD38 cells. Total RNAs was extracted after treatment. Relative real-time PCR was subjected to detect the total HBV RNAs (d) and 3.5-kb RNA levels (e), The mRNA level of β-actin was used as internal control. (f) Northern blot was applied to determine the total HBV RNAs and 3.5-kb RNA levels. The rRNA level of 28 s/18 s were used as an internal control. (g-h) 6-AN treatment decreased the level of HBV core DNA in supernatant (g) and in cells (h). The absolute quantification PCR and Southern blot (i) were performed to determine the level of HBV core DNA after 3, 6 and 9days treatment. M, marker; rcDNA, relaxed circular DNA; dsDNA, double-strand DNA; ssDNA, single-strand DNA. Results are expressed as the average of five independent experiments (n = 5 per group). The mean value ± standard error is indicated. (*P < 0.05; ^⁎⁎P < 0.01).