12 research outputs found

    <i>Vav-iCre</i> activity in non-lymphoid bone cells.

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    <p><b>(A)</b> Gating strategy. After flushing out the BM, bones were digested with collagenase and remaining cells stained for expression of the indicated markers. <b>(B)</b> EYFP expression on EC (Lin<sup>-</sup>CD45<sup>-</sup>CD31<sup>+</sup>), OB (Lin<sup>-</sup>CD45<sup>-</sup>CD31<sup>-</sup>CD51<sup>+</sup>Sca-1<sup>-</sup>), MSC (Lin<sup>-</sup>CD45<sup>-</sup>CD31<sup>-</sup>CD51<sup>+</sup>Sca-1<sup>+</sup>) and abundant Lin<sup>-</sup>CD45<sup>-</sup>CD31<sup>-</sup>CD51<sup>-</sup>Sca-1<sup>-</sup> bone cells of unknown identity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref022" target="_blank">22</a>] from (upper panels) <i>hCD2-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> (open histograms) or <i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice (shaded histograms), or from (lower panels) <i>Vav-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> (open histograms) or <i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice (shaded histograms). Numbers indicate % EYFP<sup>+</sup> cells in the respective <i>Cre</i><sup><i>+/-</i></sup> mice. Representative of three independent experiments (n = 3).</p

    <i>hCD2-iCre</i> activity in mature leukocytes.

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    <p><b>(A)</b> Gating strategy. Splenocytes were stained with the indicated AB and analyzed by FACS, gating on CD3<sup>+</sup>NK1.1<sup>-</sup> T cells, CD3<sup>-</sup>NK1.1<sup>+</sup>CD11b<sup>+</sup> mature NK cells, CD3<sup>+</sup>NK1.1<sup>+</sup> NKT cells, CD3<sup>-</sup> NK1.1<sup>-</sup>CD11c<sup>-</sup>CD11b<sup>+</sup>B220<sup>-</sup>Gr-1<sup>hi</sup> granulocytes, CD3<sup>-</sup>NK1.1<sup>-</sup>CD11c<sup>lo</sup>B220<sup>+</sup> pDC, CD3<sup>-</sup>NK1.1<sup>-</sup>CD11c<sup>hi</sup> CD11b<sup>+</sup>B220<sup>-</sup> cDC, CD3<sup>-</sup>NK1.1<sup>-</sup>B220<sup>+</sup> B cells and CD3<sup>-</sup>NK1.1<sup>-</sup>CD11c<sup>lo</sup>F4/80<sup>+</sup>CD11b<sup>+</sup> macrophages. <b>(B)</b> EYFP expression in the indicated mature leukocyte populations from <i>hCD2-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> (open histograms) and <i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice (shaded histograms). Numbers depict % EYFP<sup>+</sup> cells in the <i>hCD2-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice. Representative of three independent experiments (n = 3 for each genotype).</p

    <i>hCD2-iCre</i> activity in T, B and NK cell development.

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    <p><b>(A)</b> Thymocyte populations were identified by the gating strategy in the upper panel [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref013" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref031" target="_blank">31</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref033" target="_blank">33</a>]. Lower panel, EYFP expression in the indicated thymocyte populations from <i>hCD2-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> (open histograms) or <i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice (shaded histograms). Numbers denote % EYFP<sup>+</sup> cells within the indicated population of <i>hCD2-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice. Representative of three independent experiments (n = 3). <b>(B)</b> Upper panel, subsets of developing B cells in the BM were distinguished as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref019" target="_blank">19</a>]. Lower panel, EYFP expression in the indicated BM B cell populations from <i>hCD2-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> (open histograms) or <i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice (shaded histograms). Numbers denote % EYFP<sup>+</sup> cells within the indicated population of <i>hCD2-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice. Representative of three independent experiments (n = 3). <b>(C)</b> Upper panels, NK cell progenitors (NKP), immature (iNK) and mature (mNK) NK cells were identified using the indicated gating strategy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref018" target="_blank">18</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref029" target="_blank">29</a>]. Lower panels, EYFP expression in the indicated BM and splenic NK cell populations from (top, n = 3 per genotype) <i>hCD2-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> (open histograms) or <i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice (shaded histograms), or (bottom, n = 2 per genotype) from <i>Vav-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> (open histograms) or <i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice (shaded histograms). Numbers indicate % EYFP<sup>+</sup> cells in the respective <i>Cre</i><sup><i>+/-</i></sup> mice. Representative of three independent experiments with <i>hCD2-iCre</i> transgenic mice, and of two independent experiments with <i>Vav-iCre</i> transgenic mice.</p

    <i>Murine CD2</i> mRNA tissue expression profiles.

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    <p>Shown are ImmGen Consortium [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref053" target="_blank">53</a>] (<a href="http://www.immgen.org" target="_blank">www.immgen.org</a>) probe set 10500677 <b>(A-F)</b> and BioGPS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref054" target="_blank">54</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref055" target="_blank">55</a>] (<a href="http://www.biogps.org" target="_blank">www.biogps.org</a>) probe set 1418770_at <b>(G)</b><i>murine CD2</i> mRNA expression profiles across (A, B) key hematopoietic cell populations, (C) HSC and HPC populations, (D) B cell developmental and mature populations, (E) T cell and NKT cell developmental and mature populations, (F) DC subsets and (G) multiple hematopoietic tissues and cell types. In (G), non-hematopoietic tissues are not shown because they did not express CD2. HSC, hematopoietic stem cells; HPC, hematopoietic progenitor cells; MPP, multipotent progenitors; MDP, monocyte-DC precursors; CDP, common DC precursors; CLP, common lymphoid progenitors; Fr. A, fraction A pre-pro B cells; pro B, pro B cells; Fr. B,C, fraction B and C pro- and early pre B cells; pre B (D), fraction D late pre B cells; Fr. F, fraction F recirculating mature B cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref019" target="_blank">19</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref034" target="_blank">34</a>]; NK, NK cells; Thy, thymocyte; ETP, early thymocyte progenitor; DN3A, DN3B, DN4, CD4<sup>-</sup>CD8<sup>-</sup> thymocyte subsets; DP, CD4<sup>+</sup>CD8<sup>+</sup> thymocytes; SP, CD4<sup>+</sup> and CD8<sup>+</sup> thymocytes; T<sub>reg</sub> cells, regulatory T cells; transit. B, transitional B cells.</p

    <i>hCD2-iCre</i> activity in HSC and HPC subsets.

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    <p><b>(A)</b> Upper panels, HPC subsets were identified using the indicated gating strategy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref024" target="_blank">24</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref025" target="_blank">25</a>]. Lower panels, EYFP expression in CLP (Lin<sup>-</sup>Flk2<sup>+</sup>IL-7Rα<sup>+</sup>CD27<sup>+</sup>), MEP (Lin<sup>-</sup>IL-7Rα<sup>-</sup>c-kit<sup>+</sup>CD16/32<sup>-</sup>CD34<sup>-</sup>), CMP (Lin<sup>-</sup>IL-7Rα<sup>-</sup>c-kit<sup>+</sup>CD16/32<sup>-/lo</sup>CD34<sup>+</sup>) and GMP (Lin<sup>-</sup>IL-7Rα<sup>-</sup>c-kit<sup>+</sup>CD16/32<sup>+</sup>CD34<sup>+</sup>) from <i>hCD2-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> (open histograms) or <i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice (shaded histograms), or from <i>Vav-iCre</i><sup><i>+/-</i></sup><i>R26-stop-EYFP</i><sup><i>+/-</i></sup> (open histograms) or <i>R26-stop-EYFP</i><sup><i>+/-</i></sup> mice (shaded histograms). Representative of three independent experiments. <b>(B)</b> Upper panels, phenotypic HSC/MPP subsets were identified using the indicated gating strategy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref023" target="_blank">23</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref035" target="_blank">35</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124661#pone.0124661.ref056" target="_blank">56</a>]. Lower panels, EYFP expression in phenotypic LT-HSC (Lin<sup>-</sup>c-kit<sup>+</sup>Sca-1<sup>+</sup>CD150<sup>+</sup>CD34<sup>-</sup>CD48<sup>-</sup>Flk2<sup>-</sup>), ST-HSC1 (Lin<sup>-</sup>c-kit<sup>+</sup>Sca-1<sup>+</sup>CD150<sup>+</sup>CD34<sup>+</sup>CD48<sup>-</sup>Flk2<sup>-</sup>), ST-HSC2 (Lin<sup>-</sup>c-kit<sup>+</sup>Sca-1<sup>+</sup>CD150<sup>+</sup>CD34<sup>+</sup>CD48<sup>+</sup>Flk2<sup>-</sup>), ST-HSC3 (Lin<sup>-</sup>c-kit<sup>+</sup>Sca-1<sup>+</sup>CD150<sup>-</sup>CD34<sup>+</sup>CD48<sup>+</sup>Flk2<sup>-</sup>) and MPP (Lin<sup>-</sup>c-kit<sup>+</sup>Sca-1<sup>+</sup>CD150<sup>-</sup>CD34<sup>+</sup>CD48<sup>+</sup>Flk2<sup>+</sup>) from the mice in (A). Representative of at least two independent experiments. Numbers indicate % EYFP<sup>+</sup> cells in the respective <i>Cre</i><sup><i>+/-</i></sup> mice (n = 3 for experiments with <i>hCD2-iCre</i> transgenic mice, n = 2 for experiments with <i>Vav-iCre</i> transgenic mice).</p

    Secondary structures in 5’UTR correlate with translational repression of target genes.

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    <p>(<b>A</b>) A hairpin structure in the <i>CD69</i> 5’UTR. (<b>B</b>) Ribosome accumulation in <i>CD69</i> 5’UTR correlated with miR-17~92 family miRNA expression levels. Note that the hairpin structure co-localizes with the ribosome footprint peak in the <i>CD69</i> 5’UTR. <i>Actb</i> was used as control. (<b>C</b>) Deletion of the miR-17~92 family miRNAs shifted <i>CD69</i> mRNA from light to heavy polysomes. (<b>D</b>) Increased <i>CD69</i> expression in TKO B cells was mainly due to translation de-repression. Experiments in <b>B-D</b> were performed with 25.5h activated B cells.</p

    Quantification of miR-17~92 miRNAs and binding sites in primary B cells.

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    <p>(<b>A,B</b>) Quantitative Northern blot to determine miR-17~92 miRNA copy numbers. Indicated amounts of synthetic miR-17, miR-18a, miR-19b and miR-92 were added to naïve and activated TKO B cells before RNA extraction. The copy numbers of each miRNA subfamily were determined by Northern blot comparing WT B cells and TKO B cells with graded amounts of spike-in synthetic miRNAs, using a mixture of probes corresponding to all members of a miRNA subfamily (also see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s029" target="_blank">S7 Table</a></b>). Naïve B cells were activated with LPS and IL-4 for indicated amounts of time (h, hour). (<b>C-E</b>) Summary of miR-17~92 family miRNA copy numbers (<b>C</b>), conserved miR-17~92 family miRNA binding sites (<b>D</b>) (also see <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s030" target="_blank">S8 Table</a></b>), and ratios of conserved miR-17~92 family miRNA binding sites to miRNAs (<b>E</b>) in naïve and activated B cells.</p

    Ribosome accumulation in 5’UTR correlates with translational repression of target genes.

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    <p>(<b>A-C</b>) Ribosome accumulation in 5’UTRs of ribo-upregulated TKO targets in WT B cells (<b>A</b>), ribo-downregulated TG targets in TG B cells (<b>B</b>), but not in 5’UTRs of other targets (<b>C</b>). Ribosome occupancy in 5’UTR was normalized to the overall ribosome footprint abundance of the same gene [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.ref096" target="_blank">96</a>]. The first nucleotide of start codon is set as position 0 (grey dashed line). (<b>D</b>) Inverse correlation between ribosome occupancy in 5’UTR and the overall ribosome density on target mRNA in WT B cells. (<b>E</b>) High GC content in 5’UTRs of ribo-upregulated TKO targets.</p

    Regulation of target gene sensitivity to miRNA suppression by 5’UTR.

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    <p>(<b>A</b>) An engineered psiCheck2 vector (psiCheck-2-pd) for investigating the effect of 5’UTR and 3’UTR on reporter gene expression. TSS, transcription start site. (<b>B</b>) Experimental scheme of reporter assays in primary B cells. FACS plots show electroporation efficiency using a GFP-expressing plasmid. (<b>C,D</b>) Dual luciferase reporter assay to determine the effect of 5’UTR and 3’UTR on the reporter gene protein (luciferase activity) (<b>C</b>) and mRNA (qRT-PCR) levels (<b>D</b>). Closed and open circles indicate reporters with wild-type (wt) and mutated (mut) <i>CD69</i> 3’UTR, respectively. A comparison of renilla luciferase activity normalized to firefly luciferase activity (hRluc/Fluc) between psiCheck-2-pd containing mut and wt <i>CD69</i> 3’UTR reveals the sensitivity of the renilla luciferase mRNA (hRluc) to miR-17~92-mediated suppression. Results of normalized hRlcu/Fluc (n = 10) are from three independent experiments. Each experiment contained 3–4 replicates.</p

    Target genes exhibit different sensitivity to miR-17~92 expression level changes.

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    <p><b>(A)</b> Minimal overlap between ribo-upregulated TKO targets and ribo-downregulated TG targets. <b>(B-D)</b> The responses of ribosome density of ribo-upregulated TKO targets <b>(B)</b> and ribo-downregulated TG targets <b>(C)</b> to three miR-17~92 expression levels. Translated genes lacking miR-17~92 binding sites were used as control <b>(D)</b>. Colored bars indicate median values and error bars represent interquartile ranges. Each dot represents relative ribosome density of a unique gene. Numbers indicate p-values. <b>(E)</b> Different sensitivity of individual target genes to miR-17~92 expression level changes. Protein levels were determined by immunoblot and normalized to β-Actin (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s007" target="_blank">S7</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006623#pgen.1006623.s011" target="_blank">S11</a> Figs</b>). Target gene protein levels in WT B cells were arbitrarily set as 1.0 (n≥4). Vertical lines indicate error bars. <b>(F)</b> Relative mRNA levels of individual target genes in TKO, WT and TG B cells as determined by microarray (n = 3). <b>(G)</b> A hypothetical curve depicting target gene protein level change as a function of miRNA concentration. For a miRNA-target mRNA interaction in a given cellular context, there are a threshold level and a saturation level of miRNA concentration. miRNA suppresses target gene expression in a dose-dependent manner when miRNA concentration is between the threshold and saturation levels. Suppression does not occur when miRNA concentration is below the threshold level, while suppression reaches a maximal when miRNA concentration is above the saturation level. <b>(H)</b> The hypothetical response curves of group1, group2 and group3 target genes to miR-17~92 expression level changes. Note that the difference in amplitude for individual target genes is not depicted in this graph.</p
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