Additional Supporting Information may be found in the online version of this article.

HEP_23051_sm_SupFig1.tif777KSupplementary 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.tif515KSupplementary 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.tif422KSupplementary 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.tif5675KSupplementary 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.tif373KSupplementary 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.
HEP_23051_sm_SupTab.doc148KSupplementary Table

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