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

FilenameFormatSizeDescription
hep26537-sup-0001-suppfig1.eps1643KSupporting Figure 1: IGF2BP1 directly interacts with HULC in vivo and in vitro. (A) The interaction of IGF2BP1 with HULC ncRNA in HepG2 cells was verified by RNA-immunoprecipitation (RIP) after purification of endogenous IGF2BP1. (B) Potential IGF2BP1 binding sites (CAU) are distributed across HULC RNA. For further binding studies, HULC was split into two fragments (HULC_A (210 nt) and HULC_B (274 nt)) or left complete (HULC_FL, 484 nt). Arrows indicate PCR primer binding sites for later analysis. (C) Overview of the in vitro interaction assay applied to study IGF2BP1 binding to HULC. The same antibodies and beads as in (A) were used. An IGF2BP1 antibody or IgG control was coupled to Protein A/G beads. Beads were incubated with human recombinant IGF2BP1 and in vitro transcribed HULC fragments. Binding of IGF2BP1 towards different parts of HULC was analyzed via RT-PCR. (D) The assay efficiently and specifically purified rhIGF2BP1 as detected by western blotting (upper panel). No differential binding of IGF2BP1 to HULC could be observed using RT-PCR analysis (lower panel, expected PCR products: full-length = 110 bp, part A = 110 bp, part B = 138 bp). Each fragment bound directly and specifically to IGF2BP1. All experiments were executed at least three times. Shown are representative figures and the mean (±SEM).
hep26537-sup-0002-suppfig2.eps1608KSupporting Figure 2: Knockdown of IGF2BP1 induces while overexpression of IGF2BP1 reduces HULC expression. (A) Depletion of IGF2BP1 from HepG2 cells with two independent siRNAs resulted in a prolonged half-life of HULC. 48 h after siRNA transfection, cells were treated with alpha-amanitin (10 μg / mL f.c.) and samples were harvested at the indicated time points. The experiment was carried out in triplicates and given is the mean expression (±SEM). Gene expression was measured via qRT-PCR and HULC expression was normalized to the “0 h” time point. RNU6 expression was used as reference gene in each sample. (B) HepG2 cells were transiently overexpressed with pDEST-IGF2BP1. IGF2BP1 levels were measured using quantitative RT-PCR (left) and western blotting (right) showing efficient IGF2BP1 overexpression in HepG2 cells. PPIA was used as reference gene for PCR and GAPDH as loading control for western blotting. (C) After transient overexpression of IGF2BP1 for 72 h in HepG2 cells, HULC levels were measured showing a small but significant downregulation. The experiment was carried out in triplicates and given is the mean expression (±SEM).
hep26537-sup-0003-suppfig3.eps1108KSupporting Figure 3: Regulation of HULC expression after IGF2BP1 depletion is not mediated by miR-372. (A) HepG2 cells were transfected with control siRNA or two different IGF2BP1 siRNAs. After 72h, levels of mature miR-372 were measured using quantitative RT-PCR. No significant reduction of miR-372 could be detected despite a significant reduction of IGF2BP1. (B) HepG2, Hep3B and Huh7 cells were transfected with a miR-372 mimic or an appropriate negative control siRNA. After 24h, HULC expression was analyzed using quantitative RT-PCR. No significant downregulation of HULC could be detected despite a more than 2.000-fold overexpression of miR-372 as detected by qPCR (data not shown). All experiments were carried out in triplicates and given is the mean expression (±SEM). Gene expression levels were normalized to PPIA in each sample.
hep26537-sup-0004-suppfig4.eps1171KSupporting Figure 4: Combined depletion of IGF2BP1 and CNOT1 has no additive effect on HULC up-regulation. (A) HepG2 cells were transfected with either single IGF2BP1 or CNOT1 siRNAs or with a combination of both siRNAs (100 pmol per siRNA). After 72h, the levels of IGF2BP1 and CNOT1 were measured using quantitative RT-PCR showing significant and comparable downregulation of IGF2BP1 (upper panel) and CNOT1 (lower panel), irrespective of single or double knockdown. (B) HULC expression levels after individual or combined IGF2BP1 and CNOT1 knockdown for 72 h in HepG2 cells were significantly increased. Importantly, the combined knockdown had no additive effect on HULC expression which implicates that both proteins might be mechanistically linked. The experiment was carried out in triplicates and given is the mean expression (±SEM). Gene expression was normalized to PPIA in each sample.
hep26537-sup-0005-suppinfo.doc363K

Supporting Table 1: Patient's characteristics of expression profiling cohort

Supporting Table 2: qRT-PCR primer sequences

Supporting Table 3: siRNA sequences

Supporting Table 4: PCR primer with T7-overhang for in vitro transcription

Supporting Table 5: HULC interacting proteins

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