Processing of baculovirus-encoded DSP-PP₂₄₀ in conditioned medium requires an activity that is only secreted by Sf9 cells early but not late after infection.

<p>(A) Processing of DSP-PP₂₄₀ in conditioned medium of virus-infected Sf9 cells. Sf9 cells were infected with baculovirus containing the DSP-PP₂₄₀ cDNA. In lane 1, 3 days after infection, CM_0–3d was collected and processed for native PAGE and Stains-All staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041110#s2" target="_blank">Materials and Methods</a>. In lane 2, CM_0–3d was incubated for an additional 3 days at 28°C before processing for PAGE. M represents protein size markers. (B) Medium conditioned by infected cells 3 to 7 days after infection lacks processing activity. 3 days after infection of Sf9 cells with baculovirus containing DSP-PP_240, the medium was replaced with fresh Grace’s medium containing 10% FBS and 50 µg/ml Gentamycin and the cells were cultured for an additional 4 days. In lane 1, the resulting conditioned medium, CM_3–7d was collected and processed for native PAGE and Stains-All staining. In lane 2, CM_3–7d was incubated for an additional 3 days at 28°C before processing. M represents protein size markers. (C) Quantification of DSP-PP₂₄₀ precursor processing. Using NIH J image, we measured the image intensity of DSP-PP₂₄₀ and PP₂₄₀ bands in gels shown in panels A & B and two other similar gels. Calculation of percent processing was as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041110#s2" target="_blank">Materials and Methods</a>. Because DSP staining was very weak by Stains-All staining, total density was defined as the sum of the DSP-PP₂₄₀ and PP₂₄₀ image densities. Numbers 1–4, respectively, refer to panel A lane 1, panel A lane 2, panel B lane 1 and panel B lane 2. Error bars represent standard deviation of at least duplicate samples.</p

DSP-PP₂₄₀ is cleaved by an activity secreted into conditioned medium by uninfected Sf9 cells.

<p>CM_3–7d from virus infected cells (containing most intact DSP-PP₂₄₀), was mixed with an equal volume of 3d conditioned medium from uninfected Sf9 cells or with an equal volume of unconditioned Grace’s medium as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041110#s2" target="_blank">Materials and Methods</a>. At the times shown, medium samples were processed for native PAGE and Stains-All staining as described in Materials and Medthods. Percent processing of DSP-PP₂₄₀ was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041110#s2" target="_blank">Materials and Methods</a>. Error bars represent standard deviation of at least duplicate samples.</p

Partial Sf9 TLR1 sequence aligned with <i>Drosophila</i> TLD/TLR1 protein and <i>Homo sapiens</i> BMP1.

<p>The upper in each case represents the cloned Sf9 TLR1 sequence and lower line the test sequence. (A) The peptide sequence derived from the cloned Sf9 <i>tlr1</i> cDNA shared 65% sequence identity with <i>Drosophila melanogaster</i> TLD. (B) The peptide sequence derived from the cloned Sf9 <i>tlr1</i> cDNA shared 78% sequence identity with <i>Drosophila melanogaster</i> TLR1. (C) The peptide sequence derived from the cloned Sf9 <i>tlr1</i> cDNA shared 78% sequence identity with <i>Homo sapiens</i> BMP1.</p

Alignment of consensus sequences of tolloid proteins from <i>Culex qunquefasciatus, Aedes aegypti, Tribolium castaneum and Drosophila melanogaster.</i>

<p>Sequences of tolloid protein catalytic domains from the above four species were aligned to identify consensus sequences to be used for designing primer sequences. Consensus peptide sequences are indicated by yellow highlights. Arrows represent the positions of peptide sequences QAMRHWE and IMHYA(R/K)N(T/S) that were used to make sense primer (S) and anti-sense primer (AS). These S and AS primers encompass sequences for the Zn-binding motif (HExxHxxGFxHExxRxDRD) and for another conserved region, the Met-turn (SxMHY).</p

Mass spectrometric analyses of chymotrypsin digested mature PP₂₄₀.

<p>The location of MS/MS identified peptide sequence for PP₂₄₀ recombinant protein is labeled in red. The chymotrypsin cleavage site is located C-terminal to the underlined Tyr residue (Y).</p

Processing of DSP-PP₂₄₀ in conditioned medium is Zn-dependent.

<p>(A) Stains-All staining of DSP-PP₂₄₀ cleavage. Lane 1: CM_0–3d of virus-infected cells without further incubation. Lane 2: CM_0–3d from virus-infected cells incubated for an additional 3days. Lane 3: Same reaction as lane 2 except with addition of EGTA (22 mM). Lane 4: Same reaction as lane 2 except with addition of 1mM 1,10-phenanthroline. Lane 5: Same reaction as lane 2 except with addition of 1 mM 1,10-phenanthroline and 1 mM ZnCl₂. M represents size marker. (B) Quantification of DSP-PP₂₄₀ processing shown in panel A and two similar gels. Numbers 1–5 correspond to lanes 1–5. Error bars represent standard deviation of the mean.</p

<i>In vitro</i> response of human and mouse endothelial cells to a chemotherapeutic and an anti-angiogenesis drug.

<p>(<b>A</b> and <b>B</b>) The 48-hour cytotoxicity of cisplatin (<b>A</b>) and sunitinib (<b>B</b>) was evaluated by the SRB assay in HDMEC and MDMEC. Results are normalized against vehicle control and initial plating density. (<b>C</b> and <b>D</b>) Fold-change difference in the percentage of apoptotic cells upon treatment with cisplatin (<b>C</b>) or sunitinib (<b>D</b>) for 48 hours. Apoptosis was determined as the percentage of cells in Sub-G₀/G₁ by propidium iodide staining followed by flow cytometry. (<b>E</b> and <b>F</b>) Effect of cisplatin (<b>E</b>) or sunitinib (<b>F</b>) on the cell cycle of HDMEC and MDMEC. Results are representative of 3 independent experiments.</p

Tumor xenografts vascularized with human endothelial cells have similar microvessel density by grow faster than xenografts vascularized with mouse endothelial cells.

<p>(A) Representative photographs of HeLa tumors vascularized with either HDMEC or MDMEC. (B-D) Graphs depicting tumor growth of xenografts of HN12 (B), HeLa (C), UM-SCC-17B (D) vascularized with HDMEC or MDMEC. (E) Graph depicting tumor volume at the end of the experimental period. Asterisk depicts p<0.05. (F and G) Representative photomicrographs of tumors generated with UM-SCC-17B and HDMEC or MDMEC. Immunohistochemistry for GFP was performed to identify GFP-tagged endothelial cells. (H) Microvessel density of xenograft tumors generated with UM-SCC-17B and HDMEC or MDMEC. Results are representative of 3 independent experiments, n = 8.</p

Human and mouse dermal microvascular endothelial cells have similar angiogenic potential <i>in vitro</i> and <i>in vivo</i>.

<p>(<b>A-D</b>) Representative photomicrographs of human dermal microvascular endothelial cells (HDMEC) and mouse dermal microvascular endothelial cells (MDMEC) stably transduced with GFP under light microscopy (<b>A</b> and <b>B</b>) and fluorescence microscopy (<b>C</b> and <b>D</b>). (<b>E</b> and <b>F</b>) Representative images of capillary sprouts formed by HDMEC (<b>E</b>) and MDMEC (<b>F</b>) on 3-D type I collagen matrices. (<b>G</b> and <b>H</b>) Photomicrographs of representative fields of GFP-immunostaining (red color) used to localize the blood vessels formed by HDMEC (<b>G</b>) and MDMEC (<b>G</b>) 14 days after implantation in immunodefficient mice. (<b>I</b>) The number of microvessels in implants populated with HDMEC or MDMEC. Microvessels were counted in 10 random microscopic fields/scaffold (200×) in 6 scaffolds per group. Results are representative of 3 independent experiments, n = 6.</p

Human tumor xenografts vascularized with mouse endothelial cells is more responsive to a chemotherapeutic and an anti-angiogenesis drug.

<p>Xenograft tumors were engineered in immunodefficient mice by the co-transplantation of human tumor cells and human endothelial cells or human tumor cells and mouse endothelial cells seeded in biodegradable scaffolds measuring 6×6×1 mm. (A) Graph depicting the growth of human xenografts tumors (UM-SCC-17B cells) vascularized with HDMEC or MDMEC. As soon as we observed growth of the tumors beyond the size of the scaffold (<i>i.e.</i> when average tumor volume was 180 mm³), mice began to receive either 4 mg/kg cisplatin (i.p.) every 5 days, or 40 mg/kg sunitinib (o.r.) daily. (B) Fold change difference between the pre-treatment volume of the tumors and the volume of the same tumors after 30 days of treatment with cisplatin or sunitinib. (C) Tumor volume at the end of treatment with cisplatin or sunitinib. (D) Graph depicting the number of microvessels in tumor xenografts vascularized with HDMEC or MDMEC after treatment with cisplatin or sunitinib. Asterisk depicts P<0.05, as compared with controls. Results are representative of 3 independent experiments, n = 8.</p