These authors contributed equally to this work.
Article first published online: 1 MAY 2009
Copyright © 2009 American Association for the Study of Liver Diseases
Volume 50, Issue 4, pages 1130–1139, October 2009
How to Cite
Malz, M., Weber, A., Singer, S., Riehmer, V., Bissinger, M., Riener, M.-O., Longerich, T., Soll, C., Vogel, A., Angel, P., Schirmacher, P. and Breuhahn, K. (2009), Overexpression of far upstream element binding proteins: A mechanism regulating proliferation and migration in liver cancer cells. Hepatology, 50: 1130–1139. doi: 10.1002/hep.23051
Potential conflicts of interest: none.
The data in this study were presented in part at the 21st European Congress of Pathology (Istanbul, 2007), and the authors were awarded the George Tiniakos Award.
- Issue published online: 28 SEP 2009
- Article first published online: 1 MAY 2009
- Accepted manuscript online: 1 MAY 2009 12:00AM EST
- Manuscript Accepted: 21 APR 2009
- Manuscript Received: 21 FEB 2009
- Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Immunotherapy of Cancer
- German Research Foundation. Grant Number: Schi 273/4-3
- Dr. Mildred Scheel Foundation for Cancer Research. Grant Number: 106178
- UBS AG, Switzerland
Additional Supporting Information may be found in the online version of this article.
|HEP_23051_sm_SupFig1.tif||777K||Supplementary Figure S1: FBP-1 and FBP-2 expression in human tumor cell lines and effects on apoptosis. (A) Western immunoblotting of total protein extracts from primary human hepatocytes (PHH), hepatocellular carcinoma (HuH-7, Hep3B, PLC/PRF/5), hepatoblastoma (HepG2), squamous cell carcinoma (SCC-2), and non-small cell lung cancer cell lines (A549, NCI-H157, and Calu-6). Note that long-term exposure revealed a weak expression of FBP-1 in PHH. Tumor cell apoptosis after inhibition of FBP-1 was determined by caspase-3 assays (cleavage/activation of pro-caspase-3) and Western immunoblotting of PARP cleavage in (A) HuH-7 and (B) Hep3B cells. Equally, no effects on apoptosis were detected after inhibition of FBP-2 in both cell lines (data not shown).|
|HEP_23051_sm_SupFig2.tif||515K||Supplementary Figure S2: Inhibition of FBP-1 and FBP-2 expression by RNA interference reduces tumor cell viability or motility in HuH-7 cells. (A) Inhibitory efficiency of gene-specific siRNAs for FBP-1 and FBP-2 after transient transfection of HuH-7 cells determined by western blot analyses (B) Tumor cell viability after FBP-1 (left columns) and FBP-2 (right columns) knock down was measured 4 days after seeding (MTT-assay). (C) Tumor cell proliferation was determined based on BrdU incorporation 3 days after FBP-1 (left) and FBP-2 (right) inhibition (BrdU-ELISA). (D) HCC cell motility was determined using a 2-dimensional migration assay 3 days after FBP-1 (left) and FBP-2 (right) knock down.|
|HEP_23051_sm_SupFig3.tif||422K||Supplementary Figure S3: FBP-1 induces Stathmin/OP18 in HuH-7 cells. (A) Vector-based overexpression of FBP-1 in HuH-7 cells for four days did not affect stathmin levels. (B) siRNA-mediated knock down of FBP-2 and subsequent accumulation of FBP-1 did not induce stathmin concentrations in HuH-7 cells. (C) Relative quantification of FBP-1 (black bars) and stathmin (grey bars) transcript levels after FBP-1 knock down. (D) Transient inhibition of FBP-1 reduces stathmin concentration at the protein level in HuH-7 cells. (E) Effects of FBP-1 knock down, FCS withdrawal, and mitomycin-c treatment on proliferation and stathmin expression in HuH-7 cells. Left columns: proliferation index (BrdU-ELISA); right figure: stathmin protein levels.|
|HEP_23051_sm_SupFig4.tif||5675K||Supplementary Figure S4: FBP-1 modulates cell morphology in HuH-7 cells. Nuclear and cytoplasmic areas of tumor cells were measured after knock down of FBP-1 in HuH-7 cells. Representative cells are shown after a-tubulin staining. Untreated cells were used for calibration and nonsense siRNA-transfected cells were utilized for statistical evaluation. Bar: 30 μm.|
|HEP_23051_sm_SupFig5.tif||373K||Supplementary Figure S5: FBP-1 inhibition did not affect EZH2 expression or miR-223 concentration in HCC cells. (A) Transient transfection of FBP-1 specific siRNAs reduced FBP-1 but not EZH2 expression (anti-EZH2, Cell Signaling Technology) in Hep3B (A) and HuH-7 (B). Real-time PCR revealed that inhibition of FBP-1 did not induce miR-223 levels in Hep3B (C) and HuH-7 cells (D) (Applied Biosystems, has-miR-223, endogenous control: RNU6B). Together, these results demonstrate that FBP-1 did not affect stathmin expression through previously described effector mechanisms.|
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