Journal:

Article Title: A Family of Auxin-Conjugate Hydrolases That Contributes to Free Indole-3-Acetic Acid Levels during Arabidopsis Germination 1

doi: 10.1104/pp.104.039677

Figure Lengend Snippet: Tissue specificity of amidohydrolase gene expression. Total RNA prepared from the indicated Arabidopsis tissues was analyzed by gel-blot hybridization with antisense RNA probes prepared from ILR1, ILL1, and ILL2 or with a DNA probe that detects the 25S rRNA. Previously determined IAR3 hybridization (Davies et al., 1999) is shown for comparison.

Article Snippet: Antisense RNA probes (Riboprobe in vitro Transcription Systems; Promega, Madison, WI) were used to detect hydrolase mRNAs.

Techniques: Expressing, Western Blot, Hybridization

Journal: Molecular and Cellular Biology

Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

doi: 10.1128/MCB.00278-17

Figure Lengend Snippet: ChIRP-Seq reveals m-Meg3 enrichment at multiple m-c-Met loci. (A) Representative agarose gel image showing the specificity of the m-Meg3 ChIRP probes. RNA was isolated after m-Meg3 ChIRP from the V3 (vector) and the M5 (stable MIN6-4N cells stably expressing the m-Meg3-3 isoform which lacks exon 4) cell lines. The RNA was then used for RT-PCR. Input corresponds to the RT-PCR product using RNA isolated before m-Meg3 ChIRP-Seq. Odd and even correspond to RT-PCR using RNA after m-Meg3 ChIRP with probes located at odd and even locations on the m-Meg3 RNA. The gel image represents products of the RT-PCR performed with primers 1F/1R (flanking exon 7 and exon 8) that recognize all m-Meg3 isoforms. gapdh served as the negative control. cDNAs from three replicates of V3 and M5 ChIRP-Seq were pooled due to low yields and sequenced. (B) m-Meg3 ChIRP-PCR in a stable cell line expressing full-length m-Meg3. RNA was isolated after m-Meg3 ChIRP from the 9V (vector) and the 14M (stable MIN6-4N cells with the m-Meg3-1 isoform, which encompasses all 10 exons) cell lines. The RNA was then used for RT-PCR. Input corresponds to RT-PCR from RNA isolated before m-Meg3 ChIRP. RT-PCR was performed with primers 1F/1R (flanking exons 7 and 8) that recognize all m-Meg3 isoforms and further confirmed with the ex3F/ex4R primer pair (flanking exons 3 and 4), specific for the m-Meg3-1 isoform. gapdh served as the negative control. (C) m-Meg3 enrichment patterns at discrete m-c-Met genomic regions in different m-Meg3 stable cell lines. DNA was isolated after m-Meg3 ChIRP from two different m-Meg-3 stable MIN6-4N cell lines and their respective vector controls. The DNA was then subjected to whole-genome amplification (WGA) and subsequent purification. The purified WGA DNA was then used to set up qPCRs in duplicate with primers specific for m-c-Met genomic regions identified by m-Meg3 ChIRP-Seq, namely, the m-c-Met upstream region, the m-c-Met exon 18 region, the m-c-Met exon 20 region, and also the previously identified kb +63 enhancer. The qPCR data are represented as percent input of DNA.

Article Snippet: Antisense oligonucleotide probes for m-Meg3 ( n = 30) were designed using the online probe designer from LGC Biosearch Technologies, Petaluma, CA.

Techniques: Agarose Gel Electrophoresis, Isolation, Plasmid Preparation, Stable Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Whole Genome Amplification, Purification

Journal: Molecular and Cellular Biology

Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

doi: 10.1128/MCB.00278-17

Figure Lengend Snippet: m-Meg3 associates with PRC2 components in mouse insulinoma cell line models. (A and B) Association of m-Meg3 RNA with EZH2 and H3K27me3. RNA-ChIP in Men1 cells (Meg3 proficient) was performed using the antibodies directed toward EZH2 (A) and H3K27me3 (B). qPCR was performed on cDNA obtained from RNA isolated after RNA-ChIP. qPCR data were calculated as percent RNA input and are shown as the average from two independent biological replicates and multiple technical replicates (mean ± SD). IgG served as the negative control for the RNA-ChIP assay. (C and D) EZH2 and H3K27me3 enrichment at the m-c-Met regions identified by m-Meg3 ChIRP-Seq. ChIP assay was performed in Men1 cells using the indicated antibodies. DNA isolated after ChIP was used for qPCR, with primers specific for the m-c-Met upstream region, the +63-kb enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. EZH2 (C) and H3K27me3 (D) enrichment was calculated as percent chromatin DNA input and constitutes the average from three independent biological replicates and multiple technical replicates (mean ± SD). (E and F) Enhancer-signature histone modifications at the m-c-Met loci identified by m-Meg3 ChIRP-Seq. MIN6-4N cells were subjected to ChIP assays and qPCR analyses to detect the enrichment of H3K27Ac (E) and H3K4me1 (F) at the m-c-Met upstream region, the kb +63 enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. The data represent an average from two independent biological replicates and multiple technical replicates (mean ± SD).

Article Snippet: Antisense oligonucleotide probes for m-Meg3 ( n = 30) were designed using the online probe designer from LGC Biosearch Technologies, Petaluma, CA.

Techniques: Isolation, Negative Control

Journal: Molecular and Cellular Biology

Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

doi: 10.1128/MCB.00278-17

Figure Lengend Snippet: m-Meg3 requires PRC2 components to repress the m-c-Met transcript. (A and B) Effect of the EZH2 inhibitor GSK343 on the expression of m-Meg3 and m-c-Met transcripts. RNA was isolated from MIN6-4N and Men1 cells after vehicle or GSK343 treatments at the indicated times. The RNA isolated was used in qPCR analyses using primers specific for m-c-Met, m-Meg3-1, and m-EZH2. The qPCR transcript data shown are from representative experiments where technical replicates were set up in triplicate. ***, P ≤ 0.001; *, P ≤ 0.05; N.s., nonsignificant. (C) Depletion of H3K27me3 is a confirmatory readout for the inhibition of EZH2 methyltransferase activity. Representative Western blots for H3K27me3, using whole-cell extracts (WCEs) from MIN6-4N and Men1 cells after 7-day and 6-day treatments, respectively, with 2.5 μM GSK343 or dimethyl sulfoxide (DMSO) vehicle controls are shown. β-Actin served as the loading control.

Article Snippet: Antisense oligonucleotide probes for m-Meg3 ( n = 30) were designed using the online probe designer from LGC Biosearch Technologies, Petaluma, CA.

Techniques: Expressing, Isolation, Inhibition, Activity Assay, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

doi: 10.1128/MCB.00278-17

Figure Lengend Snippet: m-Meg3 TFO-9 regulates the m-c-Met transcript. (A) Schematic of full-length m-Meg3-1 exon structure showing the TFO-9 coordinates. Full-length m-Meg3-1 is depicted, with exons 1 to 10 numbered (top) and the sequence length in base pairs (bottom). TFO-9, a GA- and GT-rich 16-mer sequence, is shown mapping to the C-terminal portion of exon 10 in m-Meg3-1. TFO-9, spanning bp 1822 to 1838, was predicted by Triplexator to form triplexes with double-stranded DNA. (B) Effect of TFO-9 on the expression of m-Meg3 and m-c-Met transcripts. RNA was isolated at 48 h and 96 h posttransfection from MIN6-4N cells transiently transfected with TFO RNA oligonucleotides. Purified RNA converted to cDNA was subjected to qPCR analyses with primers specific for m-Meg3-1 or m-c-Met. The data represent an average from three independent experiments and multiple technical replicates (mean ± SD) *, P ≤ 0.05.

Article Snippet: Antisense oligonucleotide probes for m-Meg3 ( n = 30) were designed using the online probe designer from LGC Biosearch Technologies, Petaluma, CA.

Techniques: Sequencing, Expressing, Isolation, Transfection, Purification

Journal: Molecular and Cellular Biology

Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

doi: 10.1128/MCB.00278-17

Figure Lengend Snippet: Model depicting the mechanisms of Meg3-mediated c-Met regulation in normal pancreatic islets and tumor cells. The model presented here suggests that in the context of intact menin in normal islet beta cells, the presence of Meg3 deters the proto-oncogenic activity of c-Met by multiple mechanisms. c-Met signaling can be repressed by genomic Meg3 binding, perhaps through contact with Meg3 TFO regions and Meg3 interaction with PRC2 components. Meg3 downregulation driven by menin loss and menin-independent Meg3 loss could both favor gene-activating epigenetic changes. These epigenetic alterations in turn potentiate aberrant c-Met signaling, leading to pancreatic islet β-cell tumor formation.

Article Snippet: Antisense oligonucleotide probes for m-Meg3 ( n = 30) were designed using the online probe designer from LGC Biosearch Technologies, Petaluma, CA.

Techniques: Activity Assay, Binding Assay