Incidence of open reading frames (ORFs) in randomly generated transcripts of increasing length.

<p>Twenty thousand transcripts of varying length and random nucleotide composition were computationally generated and scanned for ORFs. The maximum ORF and transcript lengths were plotted and fitted to a logarithmic curve. The shaded regions represent incidences of randomly occurring ORFs at 1, 2, or 3 standard deviations from the mean. The red line indicates the 300 nt ORF threshold that is commonly used to distinguish protein-coding genes in transcript classification pipelines. Therefore, this plot illustrates that for transcripts longer than ∼1000 bp, such a threshold may define transcripts as protein-coding that would be expected to occur by chance. The function y = 91.Ln(x)−330, which approximates random ORF incidence according to transcript length at two standard deviations above the mean (i.e., 95% confidence interval, indicated in green), could be used to discriminate noncoding from protein-coding transcripts in a transcript-length–dependent manner.</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

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

<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

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

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

Knockdown of <i>mPINC</i> enhances differentiation of HC11 cells.

<p>(A, B) To target <i>mPINC1.0</i> and <i>mPINC1.6</i> (siPINC1.0/1.6), but not <i>DCR2</i>, siRNAs #1 and #2 were used in combination. To target all splice variants (siPINC), siRNAs #1 and #3 were used in combination. (A) Schematic showing siRNA targets of <i>mPINC</i> splice forms. (B) qPCR shows knockdown of <i>mPINC</i> 5 days post-transfection of siRNAs. Target genes were normalized to <i>Gapdh</i> and set relative to levels in the siNEG control transfected HC11 cells. (C, D) Knockdown of <i>mPNC</i> (siPINC1.0/1.6 and siPINC) splice forms increases <i>Wap</i> and <i>Ltf</i>, but not <i>Csn2</i>, expression at 24 (C) and 72 (D) hrs post-hormone induction. Target genes were normalized to <i>Gapdh</i> and set relative to levels in siNEG treated control cells. Data are presented as mean ± SEM (n = 3). (E, F) Knockdown of <i>mPINC</i> also increases dome formation compared to a control. (E) Representative brightfield images show domes following 48 hrs of hormone treatment. Scale bars represent 50 µm. (F) Domes were counted at 48 hours post-hormone treatment from nine 20× fields/experiment and data represent mean ± SEM set relative to the dome number in the siNEG treated control group (n = 6).</p

<i>mPINC</i> associates with PRC2 in the 16-day pregnant mammary gland.

<p>(A–D) RIP assays were performed with MECs purified from mammary glands at day 16 of pregnancy using antibodies to PRC2 members and its associated histone modification, H3meK27. Antibodies to MLL1 and its associated histone modification, H3meK4, were also used as negative controls. (A) RT-PCR shows <i>mPINC</i> is associated with PRC2 members and H3meK27. <i>mPINC</i> does not associate with MLL1 or H3meK4. (B–D) qPCR shows fold enrichment of <i>mPINC</i> transcript levels associated with EZH2 (B), SUZ12 (C) RpAp46 (D) and MLL1 (E) relative to <i>Gapdh</i> levels. Data represent mean ± SD (n = 3).</p

<i>mPINC</i> expression peaks in the late pregnant and early involuting gland.

<p>(A) RT-PCR shows multiple splice forms of <i>mPINC1.0</i> and <i>mPINC1.6</i> are expressed during mammary gland development. Primers designed to the extreme ends of <i>mPINC1.0</i> and <i>mPINC1.6</i> were used to amplify cDNA from mammary gland developmental stages. (w Vir.: weeks old virgin, d Preg.: days pregnancy, d Lac.: days lactation, d Inv.: days involution). PCR products were sequenced and found to be new splice forms of <i>mPINC</i>, including a new splice variant of <i>mPINC1.6</i> called <i>DCR2</i>, for deleted conserved region 2. (B) Schematic diagram of <i>mPINC</i> exonic structure. Black boxes represent exons that are always included, grey boxes are sometimes included and clear boxes are never included. Nucleotide length is indicated above each exon along with black lines that overlap the most conserved regions of the <i>PINC</i> locus, CR1 and CR2. Exon 6 sometimes has an additional 24 nucleotides at the 3′ end in the <i>mPINC1.0</i> and <i>mPINC1.6</i> splice forms. This alternative splice site does not correlate with the inclusion/exclusion of any particular exon and its function is unknown. (C) qPCR shows <i>mPINC</i> is highest during late pregnancy and early involution. Mammary glands were harvested from 3 female Balb/c mice for each stage (V: adult virgin, dP: days pregnant, dL: days lactation, dI: days involution). Target genes were normalized to <i>Actb</i> and set relative to levels in the virgin mammary gland. (D) <i>mPINC</i> expression is most abundant in the mammary gland. Tissues were harvested from three 10 week old virgin Balb/c female mice and testis, epididymis, and prostate was harvested from three 12 week old male Balb/c mice. ND indicates tissues in which <i>mPINC</i> was not detected by qPCR. Target genes were normalized to <i>Actb</i> and set relative to levels in the lung.</p

Microarray identifies potential targets of <i>mPINC</i> in HC11 cells.

<p>(A) Heat map showing transcript profiling of biological replicates of control siNEG cells, siPINC1.0/1.6, and siPINC 5 days post-siRNA transfection ( p<0.01 and fold change >1.8 in either siPINC1.0/1.6 or siPINC relative to siNEG). (B) Graph indicating the most significantly enriched gene ontology terms in the <i>mPINC</i> knockdown data set. (C) Three heat map panels depicting genes that are differentially expressed between either undifferentiated (−LH) <i>mPINC</i> knockdown cells (left panel) or differentiated (+LH) <i>mPINC</i> knockdown cells (middle panel) and differentiated (+LH) <i>mPINC</i> overexpression cells (right panel). 181 genes were found to be differentially regulated by <i>mPINC</i> (141 genes are upregulated by knockdown and downregulated by overexpression, while 40 genes are downregulated by knockdown and upregulated by overexpression at a p<0.01 and fold change >1.4, both for the overexpression and either knockdown group in the opposite direction). Genes whose expression was validated by qPCR are indicated in the heat map on the left. (D) qPCR verified differential expression of genes in <i>mPINC</i> knockdown (KD) compared to overexpression (OE) cells. qPCR was performed on biological triplicates, normalized using <i>Gapdh</i>, and shown as fold change compared to the negative control (either siNEG or LeGO-GFP).</p