Fn14•TRAIL is more potent than soluble forms of Trail and Fn14 alone or in combination.

<p>(<b>A</b>) SK-HEP-1 [A, left panel], HepG2 [A, middle panel] and Huh7 [A, right panel] HCC cell lines, as well as NKNT3 [B, left panel] and FHB [B, right panel] hepatocyte cell lines, were incubated with 0, 3, 30 or 300 ng/ml of Fn14•TRAIL, TRAIL, Fn14-Fc or combination of the later two for 48 hours. Viable cells were stained with trypan blue and counted. The results represent the mean +/- SD of three independent experiments (* <i>p</i> ≤ 0.05). (<b>B</b>) HepG2 HCC cells were incubated with 30ng/ml Fn14•TRAIL for 24 hours, in the presence or absence of anti Fn14 or anti TRAIL blocking antibodies. Treated cells were stained by Annexin V-FITC and Propidium Iodide, and counted by flow cytometer (2x10⁴ cells per sample). The results represent the mean +/- SD of two independent experiments (* p ≤ 0.05).</p

HCC and hepatocyte cell lines express TRAIL, TRAIL receptors, Fn14 and TWEAK.

<p>(<b>A</b>) The mRNA expression level of TRAIL, TRAIL receptors (DR4, DR5, DCR-1, DCR-2, OPG), Fn14 and TWEAK was determined by quantitive real-time PCR analysis. A representative experiment of three independent experiments is shown. Data are shown as average of triplicates (SD < 0.3), normalized against two endogenous control human genes, TBP and Actin-B, as calculated by Dataassist v2.0 software. (<b>B</b>,<b>C</b>) Protein expression of TRAIL, TRAIL receptors (DR4, DR5, DcR1, DcR2), Fn14 and TWEAK was determined by flow cytometeric analysis. (<b>D</b>) Fn14•TRAIL binds to HCC cells – HepG2 cells were incubated with Fn14•TRAIL, soluble TRAIL, Fn14 or the combination of the latter for 30 min at 4°c, immune-stained with PE-conjugated anti-Fn14, and analyzed by flow-cytometry. The results represent the mean +/- SD of triplicates (* <i>p</i> ≤ 0.05). </p

No hepatotoxicty is observed in Fn14•TRAIL treated mice.

<p>HepG2 Nude mice were treated daily with subcutaneous injections of 200μg of Fn14•TRAIL (n=4) or vehicle (n=4) for 8 days. One hour after last injection blood counts (<b>A</b>) liver enzymes and urea (<b>B</b>) were measured. Livers were harvested and H&E stained sections were tested (<b>C</b>). Data are presented as mean +/- S.E. </p

LDH-B does not show BIAcore binding signal with GNE.

<p>[A] Sensorgram of the interaction of GNE with 5 µM LDH-B, compared to 5 µM α-actinin 1. [B] Sensorgram showing the effect of 2 µM LDH-B on the interaction of GNE with 0.5 µM α-actinin 1 [Resp. Diff., response difference; RU, response units].</p

Fn14•TRAIL induces death of HCC cell lines in a dose dependent manner.

<p>SK-HEP-1 (<b>A</b>) HCC cells were incubated with indicated concentrations of Fn14•TRAIL for 72 [left panel], 48 and 24 hours [right panel]. (<b>B</b>,<b>C</b>) HepG2 [B, left panel] and Huh7 [B, right panel] HCC cell lines, as well as NKNT3 [C, left panel] and FHB [C, right panel] hepatocyte cell lines, were incubated with indicated concentrations of Fn14•TRAIL for 48 and 24 hours. Viable cells were stained with trypan blue and counted. Results represent the mean +/- SE of 3 independent experiments (* <i>p</i> < 0.05 ** <i>p</i> < 0.01 vs no Fn14•TRAIL). </p

Kinetics BIAcore analysis for the interaction of GNE with α-actinin 1.

<p>Kinetics values of the interactions of WT and mutant GNE proteins with α-actinin 1 as calculated using 1∶1 Langmuir model.</p

Molecular analysis of the Fn14•TRAIL.

<p>(A) The amino-acid sequence of the Fn14-TRAIL protein. The amino-acid sequence of the extra-cellular domain of human Fn14 (amino-acids 1-52 of the mature protein, marked in bold letters) are directly linked to the extra-cellular domain of human TRAIL (amino-acids 53-217 of the mature protein, non-bold letters). The underlined sequence represents the signal-peptide of the human Urokinase protein, utilized to secrete Fn14-TRAIL out of the cell and removed from the mature protein. (B) Fn14-TRAIL separated at denaturizing conditions on SDS-PAGE, Coomassie gel staining. (C) Western blot analysis with anti-TRAIL and anti-Fn14 primary antibodies. </p

LDH-B and α-actinin 1 precipitate with GNE by <i>in vitro</i> binding assay.

<p>Gelcode reagent staining of in vitro binding assay eluates resolved by SDS-PAGE. Numbers (11–13, 14–16) correspond to anion-exchange pooled fractions. A, proteins bound to GNE-bound nickel beads; B, proteins bound to GNE non-bound nickel beads; C, control proteins bound to GNE-bound nickel beads. Black arrows point to either α-actinin 1 (∼100 kDa) or to LDH-B (∼37 kDa). Both proteins were identified by MS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002477#pone-0002477-t001" target="_blank">Table 1</a>).</p

MS identification of putative GNE binding partners.

<p>Bands from gradient SDS-PAGE (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002477#pone-0002477-g002" target="_blank">Figure 2</a>) were digested by trypsin and analyzed by mass spectrometry through nano-ESI-MS-MS. For each protein, the following information is indicated: protein name, molecular mass (MW), accession number in NCBInr database, percentage of protein sequence covered by the matched and sequenced tryptic fragments, number of identified peptides out of the total number of tryptic peptides, peptides sequences, mass difference between experimental and theoretical masses, and score (aa, amino acids; expt., expected; calc., calculated).</p

WT and mutant GNE proteins interact with α-actinin 1.

<p>Sensorgrams showing the interactions of GNE (WT and mutant) proteins with different α-actinin 1 concentrations and the residual curves, showing the fitness between the model and the experimental results [Resp. Diff., response difference; RU, response units].</p