Volume 44, Issue 5, Pages 842-847 (May 2006) Effects of liver growth factors on hepadnavirus replication in chronically infected duck hepatocytes  Olivier Schorr, Christelle Borel, Christian Trepo, Fabien Zoulim, Olivier Hantz  Journal of Hepatology  Volume 44, Issue 5, Pages 842-847 (May 2006) DOI: 10.1016/j.jhep.2005.09.014 Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Growth factor effect on primary duck hepatocytes maintained in L15 medium. DNA synthesis was measured as described in Materials and Methods and is expressed as the incorporation of (3H) thymidine into acid-insoluble material per milligram of protein during 24h before harvesting (48–72h after plating and 72–96h after plating). Cells were stimulated with EGF (50ng/ml) alone or in combination with TGFβ (2ng/ml), or stimulated with TGFα (20ng/ml). Values are representative of different experiments. [This figure appears in colour on the web.] Journal of Hepatology 2006 44, 842-847DOI: (10.1016/j.jhep.2005.09.014) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Effect of cell culture on the level of different cellular cyclins. Cells were stimulated after 48 or 72h of stimulation with EGF (50ng/ml), TGFβ (2ng/ml), TGFα (10ng/ml), HGF (25ng/ml) alone or combinated. After a RT-PCR reaction with a primer for the amplification of a 462bp fragment of cyclin E (A), of a 135bp fragment of CDC 2 (B), of a 212bp fragment of cyclin A (C) and of a 521bp fragment of GAPDH (D), PCR products were separated on an agarose gel. Journal of Hepatology 2006 44, 842-847DOI: (10.1016/j.jhep.2005.09.014) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 3 (A) Southern blot analysis of protein-bound (I) and protein-free (II) DNA extracted from livers of DHBV congenitally infected embryo (1) and primary hepatocyte after 72h of culture (2) DHBV DNA was detected after hybridization with a full-length genomic DHBV probe. RC DNA, relaxed circular DNA; CCC DNA, covalently closed circular DNA. 50pg of linear DHBV DNA was used as control (C). (B) Immunofluorescence microscopy of fetal duck primary cells after 72h of culture in L15 with EGF (50ng/ml). Cells were stained with a rabbit anti-DHBc followed by anti-rabbit coupled to Texas Red and analyzed by confocal microspcopy and transmission microscopy. (III). [This figure appears in colour on the web.] Journal of Hepatology 2006 44, 842-847DOI: (10.1016/j.jhep.2005.09.014) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 4 (A) Southern blot analysis of protein-bound (I) and protein-free (II) DNA extracted from primary hepatocyte cultures of DHBV congenitally infected embryo in L15 medium after 72h of stimulation with or without (C) different growth factors: EGF (50ng/ml) and TGFβ (2ng/ml) alone or in combination. Replicative intermediates were detected after hybridization with a full-length genomic DHBV probe. RC DNA, relaxed circular DNA; CCC DNA, covalently closed circular DNA. (B) Double immunofluorescence microscopy of fetal duck primary cells after 3 days of culture in L15 with EGF (50ng/ml). Cells were first stained with an anti-BrdU/FITC antibody (I) followed by the addition of a rabbit anti-DHBc and goat anti-rabbit coupled to Texas Red (B) (II) and analyzed by confocal microspcopy and transmission microscopy. (III). [This figure appears in colour on the web.] Journal of Hepatology 2006 44, 842-847DOI: (10.1016/j.jhep.2005.09.014) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions

Fig. 5 (A) Northern blot analysis of total RNA extracted from primary hepatocyte cultures of DHBV congenitally infected embryo in L15 medium after 72 of stimulation with different growth factors: EGF (50ng/ml) and TGFβ (2ng/ml) alone or in combination. Viral transcripts were detected after hybridization with a full-length genomic DHBV probe. 3.3kb band: pregenomic RNA, C RNA (Core protein and polymerase) and pre-C/C RNA (precore protein); 2.3kb band: pre-S/S RNA (large Surface protein); 2.1kb band: S RNA (small Surface protein). (B) Quantification of viral transcrips extracted from primary hepatocyte cultures of DHBV congenitally infected embryo in L15 medium after 72 of stimulation with different growth factors: EGF (50ng/ml) and TGFβ (2ng/ml) alone or in combination. RNA extracted from liver of DHBV congenitally infected embryos was used as control. Means values are obtained from three independent experiments. [This figure appears in colour on the web.] Journal of Hepatology 2006 44, 842-847DOI: (10.1016/j.jhep.2005.09.014) Copyright © 2006 European Association for the Study of the Liver Terms and Conditions