HSA/TIMP-2 inhibits angiogenesis in vivo.

<p>(A) Representative images of blood vessels in PBS (control, upper panel) or 1 mg/dose HSA/TIMP-2-treated CAM (lower panel). (B) Quantification of CAM angiogenesis. Blood vessel number was quantified in a circular perimeter surrounding the filter disk (<i>n</i> = 10 per group). (C) Representative image of PECAM-1 blood vessel visualized by immunofluorescence staining. The vascular density of control and HSA/TIMP-2-treated tumors was determined by staining frozen sections with anti-PECAM-1 antibody (green) followed by Hoechst nuclear counterstaining (blue). Scale bar = 50 µm. (D) Quantitative measurement of vascular density in the tissue sections determined by calculating the mean pixel intensity of PECAM-1 fluorescence staining per that of Hoechst nuclear staining. (E) <i>In vivo</i> PECAM-1 protein expression detected by western blot analysis of tumor lysates. (F) Quantification of band intensity normalized by ß-actin. All quantitative data except for CAM assay represents the mean ± SEM (<i>n</i> = 4 per group). Significant difference compared to the control group: ^**<i>P</i><0.01 by Student's <i>t</i> test.</p

HSA/TIMP-2 does not inhibit MMP-2 activity.

<p>(A) <i>In vitro</i> MMP-2 inhibitory activity was measured by co-incubating with a MMP-2 NIRF probe and activated MMP-2 in the presence of TIMP-2, MMP-2 inhibitors, or HSA/TIMP-2, followed by fluorescence imaging with an IVIS-200 imaging system (<i>top</i>). The corresponding fluorescence intensity (p/s) was quantified in each sample from 3 independent experiments (<i>bottom</i>). A reaction containing NIRF and MMP-2 was used as a positive control in this experiment (lane 4). (B) Representative whole-body NIRF images. NIRF imaging of MLL tumor-bearing mice was performed at 2 h after injection of the MMP-2 NIRF probe in the presence of MMP I (<i>n</i> = 4), HSA/TIMP-2 (<i>n</i> = 5), or control PBS (<i>n</i> = 6). (C) The quantitation of fluorescent signal in the tumor. The fluorescence intensity (p/s) was quantified based on the total photon count determined from regions of interest. Each data point represents the mean ± SEM; ^#<i>P</i><0.05 <i>vs.</i> control group by two-way ANOVA followed by Bonferroni post-hoc test.</p

HSA/TIMP-2 inhibits MMP-2 expression independent of MT1-MMP expression.

<p>(A) Representative immunohistochemical images of MMP-2. The images were taken at an original magnification of 200×. (B) Quantification of MMP-2 expression determined by the percentage of brown pixels per field. (C) Representative RT-PCR images of MMP-2. HUVECs were treated with the indicated doses of HSA/TIMP-2 (lanes 2–4), and RNA was isolated 48 h after treatment. (D) Quantification of band intensity normalized by GAPDH. The quantitative data represent the mean ± SEM of duplicate samples from three independent experiments. (E) Representative images of PECAM-1, MMP-2 and MT1-MMP detected by immunofluorescence staining. Frozen sections of tumor tissues were triple-immunostained with anti-PECAM-1, anti-MMP-2 and anti-MT1-MMP followed by Hoechst nuclear (DAPI, blue) counterstaining. Scale bar = 20 µm. (F) Quantitative measurement determined by calculating the mean pixel intensity of MMP-2 (red) or MT1-MMP (green) fluorescence staining per that of Hoechst nuclear staining (blue). All quantitative data represents the mean ± SEM (<i>n</i> = 4 per group). Significant difference compared to the control group: ^**<i>P</i><0.01, ^*<i>P</i><0.05 by Student's <i>t</i> test. <i>ns</i> = non-significant.</p

HSA/TIMP-2 inhibits tumor cell proliferation but not apoptosis in prostate tumors.

<p>(A) Representative immunohistochemical images of Ki-67 and active caspase-3 immunohistochemistry. Tumor sections were obtained at 15 days after initial injection of 80 mg/kg HSA/TIMP-2 (<i>n</i> = 4 per group). The representative images were taken at an original magnification of 200×. (B) Quantification of Ki67- and active caspase-3-positive cells as expressed by the percentage of brown pixels per field. The quantitative data represent the mean ± SEM (<i>n</i> = 4 per group). Significant difference compared to the control group: ^*<i>P</i><0.05 by Student's <i>t</i> test. <i>ns</i> = non-significant.</p

HSA/TIMP-2 inhibits extracellular proteolytic activity of MMP-2.

<p>(A) Representative images of the gelatin zymographic analysis. Culture media from HUVECs treated with TIMP-2 or HSA/TIMP-2 for 72 h were analyzed on a gelatin gel. Two forms of MMP-2 (latent form, 72 kDa; activated form, 62 kDa) are presented. (B) Quantification of band intensity. Data are expressed as the percentage of band intensity normalized with respect to the proliferation level (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035710#pone.0035710.s004" target="_blank">Fig. S4</a>). The quantitative data represent the mean ± SEM of duplicate samples from three independent experiments.</p

Vertebrate orthologs of genes essential for cell migration in <i>C. elegans</i> are expressed in premigratory neural crest cells in chicken embryos.

<p>Whole mount <i>in situ</i> hybridization was performed using RNA probes corresponding to orthologs of each nematode gene (upper panel). Of the twenty-five genes examined in this study, fourteen chicken orthologs were expressed in the premigratory neural crest domain in the neural folds, as clearly shown in sections (lower panel); Crk (ced-2), Dock180 (ced-5), p21-Rac1 (<i>ced-10</i>), Chx10 (<i>ceh-10</i>), NR1I3 (<i>daf-12</i>), Fgfr1 (<i>egl-15</i>), Fgf18 (<i>egl-17</i>), Fbln2 (<i>fbl-1</i>), Frizzled10 (<i>lin-17</i>), HoxA4 (<i>lin-39</i>), HoxB6 (<i>mab-5</i>), Grb2 (<i>sem-5</i>), DCC (<i>unc-40</i>) and Kinesin (<i>vab-8</i>). arrow, plane of section; arrowhead, gene expression in neural fold.</p

Functional pathway analysis.

<p>(A) Predicted FGFR signaling pathway map showing genes obtained from our comparative analyses in red. Whole mount <i>in situ</i> hybridization using RNA probes corresponding to the genes in black was performed. (B) Table summarizing gene expression in neural crest cells. pNCC, premigratory neural crest cells: mNCC, migratory neural crest cells: -, not detected: +, detected: ++, expressed at high level. (C) Transverse sections of chick embryos showing gene expression in premigratory and/or emigrating neural crest cells at stage HH 8–9 (a–f) and in migrating neural crest cells at the midbrain level at HH stage 9–10 (a′–f′); (a, a′) frs2, (b, b′) gab1, (c, c′) pik3r2, (d, d′) shc1, (e, e′) sos1, (f, f′) sos2. Arrows indicate emigrating neural crest cells (a–d, f) or premigratory neural crest cells (e). Arrowheads indicate head mesenchymal cells (a,a′).</p

Putative chicken orthologs of nematode cell migration genes are conserved in migrating cranial neural crest cells.

<p>Chicken embryos were subjected to whole mount <i>in situ</i> hybridization using RNA probes corresponding to the vertebrate orthologs of each nematode gene. (A) Whole mount chicken embryos (upper panel) and tissue sections at the midbrain level of each embryo (lower panel) show that ten vertebrate orthologs are expressed in neural crest cells migrating from the neural tube in chicken embryos at HH stage 9–10; Crk (<i>ced-2</i>), Dock180 (<i>ced-5</i>), p21-Rac1 (<i>ced-10</i>), NR1I3 (<i>daf-12</i>), Fgfr1 (<i>egl-15</i>), Fgf18 (<i>egl-17</i>), Fbln2 (<i>fbl-1</i>), HoxB6 (<i>mab-5</i>), Grb2 (<i>sem-5</i>) and Ulk2 (<i>unc-51</i>). Arrowhead indicates migrating neural crest cells. (B) Five vertebrate orthologs are expressed in migrating neural crest cells at the hindbrain level in HH stage 11–13 embryos; Dock180 (<i>ced-5</i>), p21-Rac1 (<i>ced-10</i>), NR1I3 (<i>daf-12</i>), Fgfr1 (<i>egl-15</i>) and Kinesin (<i>vab-8</i>). Arrows indicate gene expression in migrating cranial neural crest cells in rhombomere 4 and/or rhombomere 6.</p

General and Facile Coating of Single Cells via Mild Reduction

Cell surface modification has been extensively studied to enhance the efficacy of cell therapy. Still, general accessibility and versatility are remaining challenges to meet the increasing demand for cell-based therapy. Herein, we present a facile and universal cell surface modification method that involves mild reduction of disulfide bonds in cell membrane protein to thiol groups. The reduced cells are successfully coated with biomolecules, polymers, and nanoparticles for an assortment of applications, including rapid cell assembly, in vivo cell monitoring, and localized cell-based drug delivery. No adverse effect on cellular morphology, viability, proliferation, and metabolism is observed. Furthermore, simultaneous coating with polyethylene glycol and dexamethasone-loaded nanoparticles facilitates enhanced cellular activities in mice, overcoming immune rejection

Rotarod test and PET-CT imaging.

<p>Rotarod test (A) and PET-CT imaging (B) in the sham-, vehicle-, ASA-DA-pre-ischemia-groups. Only pretreatment with 20 mg/kg ASA-DA significantly ameliorates motor activity and impaired glucose metabolism (asterisks) (n = 7 per group; *P < 0.05, significantly different from the sham-group, #P < 0.05, significantly different from the vehicle-ischemia-group). The bars indicate the means ± SD.</p