These authors contributed equally to this work.
Proteins ZNF198 and SUZ12 are down-regulated in hepatitis B virus (HBV) X protein-mediated hepatocyte transformation and in HBV replication†
Article first published online: 7 APR 2011
Copyright © 2011 American Association for the Study of Liver Diseases
Volume 53, Issue 4, pages 1137–1147, April 2011
How to Cite
Wang, W.-H., Studach, L. L. and Andrisani, O. M. (2011), Proteins ZNF198 and SUZ12 are down-regulated in hepatitis B virus (HBV) X protein-mediated hepatocyte transformation and in HBV replication. Hepatology, 53: 1137–1147. doi: 10.1002/hep.24163
Supported by National Institutes of Health (NIH) grants DK044533 and CA135192 (to O.M.A.). L.S. supported by the Bilsland's graduate student fellowship of Purdue University. The authors acknowledge support of the Purdue University Cytometry Laboratory.
- Issue published online: 7 APR 2011
- Article first published online: 7 APR 2011
- Accepted manuscript online: 10 JAN 2011 01:03PM EST
- Manuscript Accepted: 20 DEC 2010
- Manuscript Received: 28 MAY 2010
- National Institutes of Health (NIH). Grant Numbers: DK044533, CA135192
- Bilsland's graduate student fellowship of Purdue University
Additional Supporting Information may be found in the online version of this article.
|HEP_24163_sm_suppinfofig1.tif||1837K||Supporting fig. 1. Identification of genes whose protein depletion rescued pX-expressing 4pX-1 cells from DNA damage-induced apoptosis. The tetracycline-regulated pX-expressing 4pX-1 cell line (39), an immortalized, less-differentiated hepatocyte cell line (14) was used for this study. We generated a stable population of 4pX-1 cells infected with a lentiviral shRNA library. Infected 4pX-1 cells were grown for 10 days in apoptotic conditions induced by pX + doxorubicin. Cells surviving apoptosis for 10 days were propagated separately, generating individual cell clones. The shRNA insert of surviving clones was sequenced and the depleted gene identified. The most frequent genes detected by this screen were ZNF198 and SUZ12. *Topors (43) was identified by a similar screen, involving 10-day selection of infected 4pX-1 cultures with pX + doxorubicin, but without propagation of surviving cells.|
|HEP_24163_sm_suppinfofig2.tif||2306K||Supporting fig. 2. ZNF198 and SUZ12 associate with PML NBs in pX-expressing cells. A. Lysates from 4pX-1 cells grown ± pX and ± doxorubicin treatment, as indicated, were immunoprecipitated with PML antibody. IgG immunoprecipitations with lysates from 4pX-1 cells expressing pX. PML and IgG immunoprecipitates were immunoblotted for the indicated proteins. B. Confocal microscopy of 4pX-1 cells with indicated antibodies.|
|HEP_24163_sm_suppinfofig3.tif||909K||Supporting fig. 3: . Knockdown of ZNF198 and SUZ12 increases cell survival in conditions of pX-mediated apoptosis.. Lysates from indicated cell lines grown without (-) pX and in the presence of doxorubicin (0.5 μg/ml) for 6 h . Actin is loading control.|
|HEP_24163_sm_suppinfofig4.tif||1314K||Supporting fig. 4. Knockdown of ZNF198 and SUZ12 increases cell survival in conditions of pX-mediated apoptosis. A. Immunoblots of ZNF198, SUZ12, Topors and PML using lysates from clonal isolate-2 of indicated knockdown cell lines. 4pX-1 cells with stable integration of pGIPZ empty vector were constructed in parallel. B. Immunoblots of p53 using lysates from indicated clonal isolate-2. Cells were grown ± pX and doxorubicin (0.5 μg/ml) for 6 h.|
|HEP_24163_sm_suppinfofig5.tif||2119K||Supporting fig. 5: Defective DNA repair in ZNF198 and SUZ12 knockdown cells A. Comet assay of cells grown ± pX for 24 h. Cells stained with SYBR Green were analyzed by agarose gel electophoresis (13). B. Quantification of cells with DNA tailing. (250 cells counted, from three independent assays). C. Quantification of length of DNA tails using Comet Assay IV software, Perspective Instruments. D. Immunoblots of ?-H2AX using lysates from indicated cell lines grown ± pX for 24 h.|
|HEP_24163_sm_suppinfofig6.tif||828K||Supporting fig. 6. Immunoblots of ZNF198, SUZ12 and p53 using WCE isolated from the indicated cell lines after time course treatment (0-12 h) with 0.5 μg/ml doxorubicin. Actin is the loading control. The status of endogenous p53 is indicated in parenthesis.|
|HEP_24163_sm_suppinfofig7.tif||2095K||Supporting fig. 7. Immunofluorescence microscopy of endogenous PML or transfected PML-RFP protein in 4pX-1, 4pX-1-SUZ12kd, 4pX-1-ZNF198kd, and 4pX-1-PMLkd at 60X magnification. Quantification of PML NBs is from approximately 100 cells.|
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