Subset of Suz12/PRC2 target genes is activated during hepatitis B virus replication and liver carcinogenesis associated with HBV X protein†
Article first published online: 4 OCT 2012
Copyright © 2012 American Association for the Study of Liver Diseases
Volume 56, Issue 4, pages 1240–1251, October 2012
How to Cite
Studach, L. L., Menne, S., Cairo, S., Buendia, M. A., Hullinger, R. L., Lefrançois, L., Merle, P. and Andrisani, O. M. (2012), Subset of Suz12/PRC2 target genes is activated during hepatitis B virus replication and liver carcinogenesis associated with HBV X protein. Hepatology, 56: 1240–1251. doi: 10.1002/hep.25781
Potential conflict of interest: Nothing to report.
- Issue published online: 4 OCT 2012
- Article first published online: 4 OCT 2012
- Accepted manuscript online: 13 APR 2012 12:45PM EST
- Manuscript Accepted: 31 MAR 2012
- Manuscript Received: 29 JUL 2011
- National Institutes of Health (NIH). Grant Number: CA135192 and DK044533 (to O.M.A.
- “INCa, France” (to P.M. and L.L.). Grant Number: INCa Fund 19222079, RICA
Additional Supporting Information may be found in the online version of this article.
|HEP_25781_sm_SuppFig1.tif||1538K||Supporting Information Figure 1. Chromatin immunoprecipitation (ChIP) assays with Suz12 antibody or IgG as indicated in 4pX-1 cells, pX-transformed cells (14), and 4pX-1-Suz12kd cells (16). The ChIP-derived DNA was amplified by PCR with primer pairs (see Supporting information) for indicated genes and PCR products were analyzed by agarose gel electrophoresis.|
|HEP_25781_sm_SuppFig2.tif||782K||Supporting Information Figure 2. HepAD38 cells that support HBV replication following removal of tetracycline (35), were grown with (+) or without (-) tetracycline for 10 days, i.e. without (-) or with (+) HBV replication, respectively. A. 250 nM or 500 nM BI 2536 added on day 9 for 24 h (day 1) or day 8 for 48 h (day 2), respectively, as indicated, prior to cell harvesting. WCE isolated without (-) or with (+) addition of BI 2536 were immunoblotted for HBV core antigen, caspase 3 and cleaved/active caspase 3. B. Immunoblots of active Phospho-Plk1 (T210) and Plk1 using WCE isolated from HepAD38 cells grown in the presence of 250 nM BI 2536 for 2 days or 500 nM BI 2536 for 1 day, as indicated. C. 250 nM or 500 nM BI 2536 added on day 8 for 48 h (2 days) or day 9 for 24 h (1 day), respectively, as indicated, prior to isolation of total RNA. Expression of viral RNA was quantified by real-time PCR employing primer pairs described by Zhang et al, Hepatology 53, 1476-1485, 2011. Results are the average from two independent WCE preparations. Error bars indicate +/- standard error of the mean. D. Quantification of protein levels of Suz12 and Znf198 from immunoblots shown in Fig. 5B, using lysates of HepAD38 cells grown with (+) or without (-) tetracycline for 10 days and treated for 24 h prior to harvesting with 500 nM BI2536. Quantification of indicated proteins is by ImageJ software vs. actin. Results are the average from two independent WCE preparations. Error bars indicate +/- standard error of the mean.|
|HEP_25781_sm_SuppFig3.tif||539K||Supporting Information Figure 3. Analysis of microarray data from the study by Boyault et al (29). Relative induction of indicated genes is individual liver tumors with high HBV titer (H), low HBV titer (L), no HBV titer (N), and non-tumor normal liver (n).|
|HEP_25781_sm_SuppTab1.tif||403K||Supporting Information Table 1. Expression of indicated genes during oncogenic transformation of X-expressing cells. P2-P5 cultures are derived by consecutive passages of partially polyploid pX-expressing cells, isolated by live cell-sorting from the immortalized 4pX-1 cell line, as described (14). P2 and P3 cultures are not transformed, whereas P4 and P5 cultures are pX-transformed cells (14). 4pX-1-Suz12kd is a Suz12 knockdown cell line (16), used as positive control. Fold induction of indicated genes was quantified relative to expression in untransformed 4pX-1 cells. Results are from at least three independent RNA preparations used in real-time PCR reactions. Each PCR reaction was performed in triplicates and quantified relative to GAPDH used as internal control, +/- standard error of the mean.|
|HEP_25781_sm_SuppTab2.tif||845K||Supporting Information Table 2. Fold induction of indicated genes in the liver of 4-month old X monotransgenics, c-myc monotransgenics and X/c-myc bitransgenics in comparison to WT mice of the same age, quantified by real-time PCR. Results are from at least three independent RNA preparations used in real-time PCR reactions. Each PCR reaction was performed in triplicates and quantification was relative to GAPDH used as internal control, +/- standard error of the mean.|
|HEP_25781_sm_SuppTab3.tif||1504K||Supporting Information Table 3. Fold induction of the proliferation gene cluster and Suz12-repressed genes quantified in tumor vs peri-tumoral paired tissues from liver of individual WHV-infected animals. The woodchuck identification number is from the study by Jacob et al (39). Results are from at least three independent RNA preparations used in real-time PCR reactions. Each PCR reaction was performed in triplicates and quantification was relative to GAPDH used as internal control, +/- standard error of the mean.|
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