SEARCH

SEARCH BY CITATION

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

FilenameFormatSizeDescription
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

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.