Additional file 2: of Patient-derived multicellular tumor spheroids towards optimized treatment for patients with hepatocellular carcinoma

Figure S1. Wound healing assay of primary HCC cells. Wound healing assay. (A) A wound was introduced after cells reached 80-90% confluence. Cell migration was monitored under microscopy for the indicated time. Scale bar = 500 μm. (B) Cell growth was monitored for 7 days, and was quantitated of migration rate which is represented by % of control. Figure S2. Karyoviews of AMC-H1 and AMC-H2. The blue bar indicates gain, the red bar indicates loss, and the purple bar indicates LOH. Figure S3. Characterization of various primary HCC cells. (A) Capacity of various primary HCC to form tumor spheroids and MCTS. (B) Albumin and HepPar-1 immunostaining to examine the cellular origins of primary HCC cells. (C) AFP and albumin mRNA expression levels in primary HCC cells. (PDF 790 kb

Additional file 4: of Patient-derived multicellular tumor spheroids towards optimized treatment for patients with hepatocellular carcinoma

Table S3. Chromosome gains detected by SNPs array in AMC-H1 and AMC-H2. (DOCX 26 kb

Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag

<div><p>Human vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. <i>Escherichia coli</i> is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in <i>E</i>. <i>coli</i> has proven difficult because the <i>E</i>. <i>coli</i>-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF). We created seven N-terminal fusion tag constructs with hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), and tested each construct for soluble overexpression in <i>E</i>. <i>coli</i>. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1⁺ mesodermal precursor cell (MPC) differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in <i>E</i>. <i>coli</i> have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in <i>E</i>. <i>coli</i>.</p></div

Analysis of hVEGF purification from <i>E</i>. <i>coli</i>.

<p>(A) Schematic overview of hVEGF purification. (B) His6-MBP-hVEGF was purified from <i>E</i>. <i>coli</i> by chromatography. Lane 5 shows that the MBP tag was almost completely cleaved. M, molecular weight marker; lane 1, total cell extract before IPTG induction as negative control; lane 2, total cell extract with IPTG induction; lane 3, soluble fraction after cell sonication; lane 4, His6-MBP-hVEGF fusion protein purified using MBP column (62.9 kDa); lane 5, His6-MBP tag cleavage with TEV protease (28.6 kDa): His6-MBP (43.9 kDa) and hVEGF (19 kDa); lane 6 and 7, purified hVEGF using Heparin column: final hVEGF product under reducing and non-reducing conditions, respectively. The arrows indicate positions of hVEGF as monomer (19 kDa), dimer (38 kDa) and oligomers (≥100 kDa). (C) SDS-PAGE (lanes 1–3) of reduced and non-reduced samplesand Western blot analysis (lanes 4–6) with anti-hVEGF. The arrows indicate the band signals of hVEGF in fusion with MBP tag or in final product. M, molecular weight marker; lane 1 and 4, total cell extract with IPTG induction; lane 2 and 5, final hVEGF product under reducing condition; lane 3 and 6, final hVEGF product under non-reducing condition.</p

Expression analysis of tagged hVEGF in <i>E</i>. <i>coli</i> Origami 2 (DE3) by SDS-PAGE.

<p>Expression of full-length hVEGF was induced by 0.5 mM IPTG at 37°C (A) and 18°C (B). Arrows indicate the target fusion proteins. His6, hexa (poly) histidine; Trx, thioredoxin; GST, glutathione-S-transferase; PDIb'a', b'a' domain of full-length human PDI; MBP, maltose-binding protein; NusA, N-utilization substance protein A; PDI, full-length human PDI; M, molecular weight marker; C, total cell protein before IPTG induction as negative control; I, total cell protein after IPTG induction; P, pellet fraction after cell sonication; S, soluble fraction after cell sonication.</p

Purification of hVEGF from MBP-hVEGF expressed in <i>E</i>. <i>coli</i> at 18°C^a.

<p>Purification of hVEGF from MBP-hVEGF expressed in <i>E</i>. <i>coli</i> at 18°C<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156296#t002fn001" target="_blank">^a</a>.</p

Schematic representation of the domain structure and generation of the MBP-hVEGF construct.

<p>(A) Vector map of pHMGWA-hVEGF using the gateway cloning method. BP reaction was done by recombination between attB substrate (attB1-hVEGF-attB2 sequence) and attP substrate (pDONOR207). LR reaction was done by recombination between attL substrate (pENTR-hVEGF) and attR substrate (pDEST-Tag). att, site-specific attachment (att) sites; TEVrs-hVEGF: gene sequence encodes for TEV recognition site and hVEGF protein. Expression of the fusion proteins in <i>E</i>. <i>coli</i> is driven by the IPTG-inducible T7 promoter with ampicillin as the selection marker.(B) Schematic structure of the seven fusion proteins His6-, Trx-, GST-, MBP-, NusA-, PDIb'a'- and PDI-hVEGF (total size). The arrow indicates the TEV protease cleavage site.</p

Expression and solubility level of hVEGF with the seven different tags^a.

<p>Expression and solubility level of hVEGF with the seven different tags<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156296#t001fn001" target="_blank">^a</a>.</p

<i>In vitro</i> biological activity of purified hVEGF on differentiation of Flk1⁺ MPCs to CD31⁺/CD144⁺ ECs.

<p>FACS analysis of Flk1⁺ MPCs incubated with various concentrations of purified hVEGF from <i>E</i>. <i>coli</i> and mammalian cells for 48 h. (A) The density plots showed the percentage of CD31⁺/CD144⁺ expressing ECs differentiated from Flk1⁺ MPCs. (B) The density plots represented the population of CD144⁺ and CD140b⁺ expressing cells. hVEGF treatment markedly increased the number of CD144⁺ expressing cells, whereas CD140b⁺ expressing cells were reduced. (C) Percentage of CD31⁺/CD144⁺ expressing EC population. Each sample was performed in at least three independent replicates.</p