Expression cloning of human CD2 by using CD58-displaying BV as the probe.

<p>(A) Enrichment of human CD2-positive cells from BaF/3 cells transfected with a human T cell cDNA library. The staining of cells with an FITC-labeled anti humanCD2 monoclonal antibody is shown. The thin line indicates cells incubated without anti-CD2 antibody. (B) Binding of anti-human CD2 antibody and CD58-displaying BV to BaF/3 cells isolated by magnetic sorting with CD58-BV. After the 3rd magnetic sorting and subcloning, cells were stained with an FITC-labeled anti-human CD2 monoclonal antibody and CD58-BV plus biotinylated anti-gp64 antibody and PE-streptavidin. Staining of a representative of three clones is shown.</p

Expression of heterologous membrane proteins in BV fraction confirmed by Western blot.

<p>Ten micrograms of each of the BV samples expressing CD2 or its ligand CD58 (A), CD40 or CD40L (B), and GITR or GITRL (C) were loaded in each lane. The blotted membranes were immuno-stained with either anti-FLAG or anti-HA antibodies according to the attached tag as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004024#s4" target="_blank">Materials and Methods</a>. The positions of the molecular mass marker proteins are indicated on the <i>left</i>.</p

Specific binding of ligand-displaying BV to cells expressing receptor.

<p>(A) Schematic view. X corresponds to CD2 (for B), CD40 (for C), or GITR (for D). Y corresponds to CD58 (for B), CD40L (for C), or GITRL (for D). (B) CD58-displaying BV binds to CD2-positive (top), but not to CD2-negative Jurkat cells (bottom). Left: binding of an anti-CD2 monoclonal antibody (clone RPA-2.10) plus a PE-anti mouse Ig-κ chain antibody. Right: binding of CD58-displaying BV plus an anti-gp64 antibody (clone A0505A) and a PE-anti mouse Ig-κ chain antibody. The thin line indicates cells incubated with the 2nd antibody only. (C) CD40-dependent binding of CD40L-displaying BV to mouse splenic B cells. BALB/c mouse splenocytes were incubated with CD40L-displaying BV and a biotinylated anti-gp64 monoclonal antibody (clone B8147A), and then were stained with FITC-labeled streptavidin and a PE-labeled anti-B220 antibody. CD40L-BV binding to the B cells (shown in the center lower panel) was blocked by pre-incubation with an anti-mouse CD40 monoclonal antibody (clone HM40-3) (the right upper panel) but not by control hamster IgG (right lower panel). (D) Binding of GITRL-displaying BV to GITR-positive cells. GITR-expressing mouse T cell hybridoma 18.3.5 cells were incubated with GITRL-displaying BV (right) or wild-type BV (left). Binding of BV was detected with a biotinylated anti-gp64 monoclonal antibody B8147A and PE-streptavidin. The thin line indicates cells incubated without BV.</p

Detection of specific interaction between CD2 and CD58 individually displayed on BV.

<p>(A) Schematic view of the ELISA system. Details are described in the text. X and Y correspond to CD2 and CD58 (or vice versa for C) respectively. (B) Left panel; binding of CD58-BV to immobilized CD2-BV (filled circles) or wild-type BV (open circles). Right panel: Blocking of CD58-BV binding to the plate-bound CD2-BV by pre-incubation of wells with an anti-CD2 antibody (filled circles) or control mouse IgG1 (open circles). (C) Left panel: binding of CD2-BV to the immobilized CD58-BV (filled circles) or wild-type BV (open circles). Right panel: blocking of CD2-BV binding of the plate-bound CD58-BV by pre-incubation of wells with an anti-CD58 antibody (filled circles) or control mouse IgG2a (open circles). Each well was coated with 0.5 µg of BV. Each data point represents detection in triplicate (error bar, 1 s.d.).</p

Detection of specific interaction between CD40 and CD40L or GITR and GITRL displayed on BV.

<p>(A) Left panel: binding of CD40L-BV to the immobilized CD40-BV. Wells were coated with 0.25 µg/well of CD40-BV or wild-type BV. Binding was detected by using a biotinylated anti-mouse CD40L monoclonal antibody (clone MR1) and HRP-streptavidin. Right panel: binding of CD40-BV to the immobilized CD40L-BV. Wells were coated with 1 µg/well of CD40L-BV or wild-type BV. Binding was detected by using an anti-mouse CD40 monoclonal antibody (clone 3/23) and HRP-anti rat IgG+IgM. (B) Left panel: binding of GITRL-BV to the immobilized GITR-BV. Wells were coated with 1 µg/well of GITR-BV or wild-type BV. Binding was detected by using an anti-mouse GITRL monoclonal antibody (clone YGL386) and HRP-anti rat IgG+IgM. Right panel: binding of GITR-BV to the immobilized GITRL-BV. Wells were coated with 1 µg/well of GITRL-BV or wild-type BV. Binding was detected by using an anti-mouse GITR monoclonal antibody (clone DTA-1) and HRP-anti rat IgG+IgM. Filled circles indicate binding to BV displaying respective receptors (or ligands). Open circles indicate binding to wild-type BV.</p

The Molecular Profiles of Neural Stem Cell Niche in the Adult Subventricular Zone

<div><p>Neural stem cells (NSCs) reside in a unique microenvironment called the neurogenic niche and generate functional new neurons. The neurogenic niche contains several distinct types of cells and interacts with the NSCs in the subventricular zone (SVZ) of the lateral ventricle. While several molecules produced by the niche cells have been identified to regulate adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of the genetic inducible fate mapping system (GIFM) and transgenic mice to isolate the SVZ niche cells including NSCs, transit-amplifying progenitors (TAPs), astrocytes, ependymal cells, and vascular endothelial cells. From the isolated cells and microdissected choroid plexus, we obtained the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method. We identified a total of 151 genes encoding secretory or membrane proteins. In addition, we obtained the potential SMEP of NSCs using cDNA microarray technology. Through the combination of multiple screening approaches, we identified a number of candidate genes with a potential relevance for regulating the NSC behaviors, which provide new insight into the nature of neurogenic niche signals.</p> </div

Complete blood cell count of Robo4-mutant mice.

<p>Data are mean +/− S.D. (n = 5 for each genotype).</p

Long-term repopulation assay of HSCs.

<p>One hundred CD34⁻KSL cells from WT or Robo4-mutant mice (Ly5.2) were transplanted into lethally irradiated recipients (Ly5.1) with 2×10⁵ competitors. Percentage of donor chimerism (Ly5.2) in peripheral blood was examined at the indicated time points. Data are shown as mean +/− S.D. (n = 6 for WT and H, n = 8 for KO). *p<0.05. WT; wild-type, H; heterozygous, KO; knockout.</p

BM recoveries after myelosuppression.

<p>WT or Robo4^−/− mice were treated with intraperitoneal injection of 5-FU (150 mg/kg). WBC (A) and platelet (PLT) (B) count was monitored at the indicated time points. Data are presented as mean+/−S.D. (n = 5).</p

Spine Formation Pattern of Adult-Born Neurons Is Differentially Modulated by the Induction Timing and Location of Hippocampal Plasticity

<div><p>In the adult hippocampus dentate gyrus (DG), newly born neurons are functionally integrated into existing circuits and play important roles in hippocampus-dependent memory. However, it remains unclear how neural plasticity regulates the integration pattern of new neurons into preexisting circuits. Because dendritic spines are major postsynaptic sites for excitatory inputs, spines of new neurons were visualized by retrovirus-mediated labeling to evaluate integration. Long-term potentiation (LTP) was induced at 12, 16, or 21 days postinfection (dpi), at which time new neurons have no, few, or many spines, respectively. The spine expression patterns were investigated at one or two weeks after LTP induction. Induction at 12 dpi increased later spinogenesis, although the new neurons at 12 dpi didn’t respond to the stimulus for LTP induction. Induction at 21 dpi transiently mediated spine enlargement. Surprisingly, LTP induction at 16 dpi reduced the spine density of new neurons. All LTP-mediated changes specifically appeared within the LTP–induced layer. Therefore, neural plasticity differentially regulates the integration of new neurons into the activated circuit, dependent on their developmental stage. Consequently, new neurons at different developmental stages may play distinct roles in processing the acquired information by modulating the connectivity of activated circuits via their integration.</p> </div