Ptprj mRNA expression in murine tissues.

<p>(A). qPCR of <i>Ptprj</i> from RNA extracted from mouse tissues. (B) qPCR of <i>Ptprj</i> from RNA from pre-B lymphoid cell line WEHI-231, osteoclast-like cell line (RAW 264.7, C4), TEPM, macrophage-like cell line (RAW 264.7), BMM, myeloid cell line M1 and fibroblasts (NIH3T3 and mouse embryonic). (C). Immunohistochemistry of cell-specific expression of PTPRJ in mouse spleen sections. Sections were immunostained for CD148 (A1, B1) or F4/80 (A2, B2) and with CD148 (C1) and F4/80 (C2) isotype control antibodies. All sections were counterstained with haematoxylin. RP, red pulp; WP, white pulp. Original magnification: x100 (A), x200 (B, C). Bar, 100µm.</p

Regulation of <i>Ptprj</i> expression in mouse macrophages by proinflammatory stimuli.

<p>A–C: Regulation of <i>Ptprj</i> expression by CSF-1 and LPS in mouse bone marrow-derived macrophages (BMMs). BMMs were maintained overnight in the presence or absence of CSF-1 (1×10⁴ U/mL) before treatment with LPS (10 ng/mL) (A, B). RAW 264.7 cells were maintained overnight in the absence of CSF-1 before treatment with LPS (10 ng/mL) (C). <i>Ptprj</i> (A, C) and <i>c-fms</i> (B) expression profiles were assessed by quantitative real-time PCR. Profiles are representative of two independent experiments. D: Regulation of <i>Ptprj</i> expression by CSF-1 and LPS in mouse thioglycollate-elicited peritoneal macrophages (TEPMs). TEPMs were maintained overnight in the presence or absence of CSF-1 (1×10⁴ U/mL) before treatment with LPS (10 ng/mL). <i>Ptprj</i> expression profile was assessed by quantitative real-time PCR. E: IFNγ treatment of bone marrow derived macrophages suppresses the LPS mediated induction of <i>ptprj</i>. BMMs were maintained overnight in the presence of CSF-1 (1×10⁴ U/mL) and presence or absence of IFNγ (500 pg/mL) before treatment with LPS (10 ng/mL). RNA was extracted at each time point and used for the synthesis of cDNA. <i>Ptprj</i> expression profile was assessed by quantitative real-time PCR. Datapoints (+/− SD) represent the average of triplicate samples each from triplicate independent experiments. Significance values were determined by one-way analysis of variance (ANOVA). *denotes p<0.05; **denotes p<0.005; n = 3. F: Regulation of <i>PTPRJ</i> protein in response to LPS, CpG DNA and CSF-1. BMMs were maintained overnight in the presence or absence of CSF-1 (1×10⁴ U/mL) before treatment with LPS (10 ng/mL) [top panel] or CpG DNA (0.1 µM) [bottom panel]. Protein lysates were separated by SDS-PAGE, transferred to PVDF membranes and immunoblotted for PTPRJ. The membrane was then stripped, and reprobed for total Akt as a loading control. Profiles are representative of two independent experiments.</p

Characterisation of <i>Ptprj-as1.</i>

<p>A–B: <i>Ptprj-as1</i> maps to the reverse strand within the boundaries of the murine <i>Ptprj</i> gene. Comparison of the mouse Ptprj (A) and human PTPRJ (B) loci. Protein coding transcript isoforms of Ptprj/PTPRJ are shown in red and long noncoding transcripts are shown in blue. Arrows indicate the direction of transcription. The human microRNA miR-3161 is shown in green. Position of PCR primers used for qRT-PCR for mouse Ptprj-as1 are indicated. C-D: Mapping (C) and expression (D) of a splice variant of murine <i>Ptprj-as1</i> in brain, kidney and testis. E: Predicted secondary structure of <i>Ptprj-as1</i> splice variant.</p

Expression of <i>Ptprj-as1</i> in murine tissues and in response to TRL ligands.

<p>A: Expression of <i>Ptprj-as1</i> in murine tissues. B, C: <i>Ptprj</i> and <i>Ptprj-as1</i> mRNA expression in BMMs in response to LPS (B) or Pam3Cys (C). mRNA expression was quantified by qRT-PCR and expressed as fold change compared with untreated (0h). Plots represent mean fold change +/− SD; n = 3. Significance values were determined by one-way analysis of variance (ANOVA). *denotes p<0.05; **denotes p<0.005; n = 3. *denotes p<0.05; **denotes p<0.005; n = 3.</p

<i>Ptprj</i> expression in response to LPS in human mononuclear phagocytic cells.

<p>THP-1 cells were maintained for 24 hours in the presence or absence of PMA (10⁻⁷ M) to induce differentiation, before treatment with LPS (10 ng/mL) (A, B). Human dendritic cells were treated with LPS (10 ng/mL) over a time course (C, D). <i>Ptprj</i> (A, C) and <i>c-fms</i> (B, D) expression profiles were assessed by quantitative real-time PCR. Datapoints (+/− SD) represent the average of triplicate samples. Significance values were determined by one-way analysis of variance (ANOVA). *denotes p<0.05; **denotes p<0.005; n = 3. *denotes p<0.05; **denotes p<0.005; n = 3.</p

<i>In vivo</i> DMS footprinting of the <i>Csf1r</i> promoter and FIRE in stimulated BMM.

<p>Macrophages were differentiated from mouse bone marrow under the influence of CSF-1 (+) and subjected to DMS footprinting after either starving cells of CSF-1 (−) or restimulation with CSF-1 (<b>A</b>), LPS (<b>B</b>), or CSF-1 & LPS (<b>C</b>) for the indicated time points. G: Maxam-Gilbert reaction followed by LM-PCR with purified genomic DNA. ES: <i>In vivo</i> footprinting performed with ES cells. Putative transcription factor binding sites in chromatin showing alterations in methylation compared to a Maxam-Gilbert G-reaction of naked genomic DNA are shown as vertical bars on the right hand side of the gel images. Nucleotide positions relative to the ATG start are designated by numbers on the left. Macrophage specific footprints are indicated as circles (black: enhancement, white: inhibition) while LPS responsive footprints are indicated as squares (black: enhancement, white: inhibition). L-shaped arrows are the position of antisense RNA start sites at FIRE.</p

Effect of CSF-1 and LPS on <i>Csf1r</i> primary and antisense transcripts.

<p>(<b>A</b>) Macrophages were differentiated from mouse bone marrow under the influence of CSF-1. Cells were starved of CSF-1 for 24 hours, and then re-stimulated with a combination of CSF-1 and LPS. Primary RNA levels containing intronic sequences were measured by Real-Time PCR assays using primers downstream of the promoter, FIRE or intron 3. Normalisation was performed using rRNA-specific primers. Error bars represent the mean value of triplicate PCRs. (<b>B</b>) Cells and treatments were identical to (A). ChIP assays measuring the recruitment of Serine 5 phosphorylated RNA Pol II using primers covering the indicated cis-regulatory elements. Prom – 1.5 kb refers to a region upstream of the transcription start site which served as an internal negative control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054935#pone.0054935-Tagoh3" target="_blank">[29]</a>. Values were normalised against an internal negative control as described in materials and methods. Measurements are representative of at least two independent experiments and error bars represent the mean value of three different measurements. (<b>C</b>) Primers were produced that contain sequence from the positive strand of <i>Csf1r</i> upstream of the FIRE region within intron 2 (Asp1 or Asp2). These primers prime negative strand transcripts by reverse transcriptase reaction (+). DNAse treated RNA was used for the reaction and as a control RNA was primed in the absence of reverse transcriptase (−). cDNA products were detected by PCR with nested forward (NF1, NF2, or NF3) and reverse (NR) primers. (<b>D</b>) Cells and treatments were identical to (A). Antisense RNA-expression was assayed by real-time quantitative PCR exactly as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054935#pone.0054935-Tagoh2" target="_blank">[20]</a>.</p

The enhancer activity of FIRE is orientation dependent and requires the transcription start sites.

<p>(<b>A</b>) Schematic of FIRE constructs: the entire <i>Csf1r</i> regulatory region plasmid (pGL-7.2 fms), pGL-7.2 fms with FIRE subcloned into the reverse orientation (pGL-7.2 fms FIRE-), pGL-7.2 fms with FIRE deleted (pGL-7.2 fms ΔFIRE), and pGL-7.2 fms with intron 2 deleted leaving 3.5 Kb of the <i>Csf1r</i> promoter (pGL2-3.5 fms). (<b>B</b>) RAW264.7 cells were transfected with pGL-7.2 fms, pGL-7.2 fms FIRE-, or pGL-7.2 fms ΔFIRE constructs and luciferase activity was assessed. Data is shown as a percentage of pGL2-7.2 fms (100%) and error bars represent the SEM. Statistically significant differences versus pGL-7.2 fms are indicated (t-test; ***p<0.001). (<b>C</b>) RAW264.7 cells were transfected with pGL2-3.5 fms, pGL-7.2 fms, or pGL-7.2 fms FIRE- constructs. Following treatment with LPS, luciferase activity was assessed. Data for each construct are shown normalised to the same construct untreated and error bars represent the SEM. Statistically significant differences between the LPS treated construct versus the same construct untreated are indicated (t-test; **p<0.01). (<b>D</b>) RAW264.7 cells were transfected with linearized pGL-7.2 fms or a linearized pGL-7.2 fms construct containing one of the four 6 bp deletions in FIRE shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054935#pone-0054935-g004" target="_blank">Figure 4D</a>. Forty-eight hours post transfection, luciferase activity was assessed. The data represents six separate experiments performed in triplicate and error bars represent the SEM. Data are shown as a percentage of pGL2-7.2 fms (100%). Statistically significant differences versus pGL-7.2 fms are indicated (t-test; *** p<0.001, ** p<0.01, and * p<0.05).</p

Number of significant differentially abundant OTUs identified using DESeq2 (padj < 0.05), for all group comparisons.

<p>Number of significant differentially abundant OTUs identified using DESeq2 (padj < 0.05), for all group comparisons.</p

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京都大学0048新制・課程博士博士(医学)甲第12924号医博第3084号新制||医||946(附属図書館)UT51-2007-H197京都大学大学院医学研究科分子医学系専攻(主査)教授 篠原 隆司, 教授 山中 伸弥, 教授 瀬原 淳子学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA