12 research outputs found

    Engraftment and growth of U266<sup>luciferase</sup> cells as monitored by BLI, serum paraprotein and MRI. A.

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    <p>(i) Dorsal and (ii) ventral BLI acquired from IVIS over weeks 3–7 post-inoculation. <b>B.</b> Quantitative measurement of radiance from BLI. Radiance reflects the intensity of luciferase luminescence and therefore number of luciferase-tagged cells present. Results show that radiance increases in a time-dependent manner over the course of the experiment and that a significant increase in radiance occurs over weeks 5–7 (p<0.05, 1-way ANOVA with Bonferroni post-test). <b>C.</b> Paraprotein levels in the serum increases in a time dependent manner and correlates with the increase seen in BLI. MRI-derived tumour volumes determined at approximately weeks 5 and 10 confirmed tumour progression seen with BLI.</p

    BLI and serum paraprotein changes in response to therapy.

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    <p><b>A.</b> (i) Pre-treatment and (ii) post-treatment BLI of mice at weeks 5 and 9. <b>B.</b> Quantitative measurement of radiance from BLI. (i) No significant difference in radiance between treatment groups was seen at the start of the treatment schedule. (ii) Post-treatment radiance levels revealed a significant attenuation of tumour spread by both BZB and tosedostat (p<0.05, 1-way ANOVA with Bonferroni post-test). <b>C.</b> Paraprotein levels during treatment schedule. Positive control mice showed an exponential increase in serum levels of Igλ over 9 weeks. In comparison, both treatment groups did not exhibit the same increase, with significantly lower levels by the end of treatment (p<0.05).</p

    CD138 expression changes in response to therapy.

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    <p><b>A.</b> Percentage of CD138<sup>+</sup> human myeloma cells measured by flow cytometry in bone aspirates of mice (n = 3), showing a significantly lower percentage of positive cells in both tibias and spine of mice in the two treatment groups than in untreated mice (p<0.05, 2-way ANOVA with Bonferroni post-test). No CD138<sup>+</sup> cells were observed in the organs of any of the mice. <b>B.</b> Histological analysis of sections from the tibias of mice from each group showed distinct differences. (i) Sections from healthy mice displayed classical architecture, with no CD138<sup>+</sup> cells. (ii) In comparison, sections from untreated myeloma mice showed a high infiltration of CD138<sup>+</sup> cells with loss of normal architecture. (iii) Treatment of mice with BZB resulted in the return of normal architecture and loss of CD138<sup>+</sup> cells. (iv) A similar result was observed in mice treated with tosedostat, but with occasional scattered CD138<sup>+</sup> cells.</p

    MRI changes in response to therapy.

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    <p>MRI-derived tumour volumes. <b>A.</b> Tumour was identified as a hyperintense signal enclosed within the cortical bone on T<sub>2</sub>-weighted images. MRI images showed a reduction in signal intensity in both treatment groups compared to positive control in both the tibia (T) and femur (F). <b>B.</b> Tumour volume was quantified from regions of interest drawn on the periphery of the hyperintense signal. Data are mean ± SEM, n≥6. Both BZB and tosedostat (TDT) treatment resulted in a significantly lower tumour volume compared to control (p<0.05, 1-way ANOVA with Bonferroni post-test). In addition, there was no significant difference in tumour volume between the BZB treated and negative control group.</p

    REIIBP interacts with the SMN complex in myeloma t(4;14) cells.

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    <p>A) Identification of REIIBP binding partners. REIIBP protein partners were pulled down by a tandem affinity purification approach. To isolate a protein fraction with a low level of background contaminants, the lysates from H929::REIIBP and H929 (control sample) were passed through two different chromatography columns, an anti-Flag resin and Nickel resin. The purified populations were run on SDS acrylamide gel and stained with Coomassie blue. Discrete bands were cut from the gel and analysed by mass spectrometry. The number on the right indicates the position of the respective molecular weights in KDa. The positions for the proteins identified by Mass spectrometry are shown by arrows. B) Western blot analysis on Flag IP fractions. The names of the respective antibodies used for staining are shown. Molecular weight is shown. C–D) Endogenous REIIBP interacts with the SMN complex in myeloma cells. Co-IPs of GEMIN5 (C) and SMN (D) in t(4;14) untransduced myeloma cells. GEMIN5 and SMN were immuno precipitated from H929 cell lysates using specific antibodies. An IP with normal mouse IgG antibody was used as control. The IPs were resolved in a SDS acrylamide gel and blotted for the indicated antibodies. The black star shows the position for the heavy chains of the antibodies used in the Co-IP. Please note MMSET II in H929 cells has a shorter molecular weight than canonical 150 KDa.</p

    A Novel Functional Role for <i>MMSET</i> in RNA Processing Based on the Link Between the REIIBP Isoform and Its Interaction with the SMN Complex

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    <div><p>The chromosomal translocation t(4;14) deregulates <i>MMSET</i> (<i>WHSC1</i>/<i>NSD2</i>) expression and is a poor prognostic factor in multiple myeloma (MM). <i>MMSET</i> encodes two major protein isoforms. We have characterized the role of the shorter isoform (REIIBP) in myeloma cells and identified a clear and novel interaction of REIIBP with members of the SMN (survival of motor neuron) complex that directly affects the assembly of the spliceosomal ribonucleic particles. Using RNA-seq we show that REIIBP influences the RNA splicing pattern of the cell. This new discovery provides novel insights into the understanding of MM pathology, and potential new leads for therapeutic targeting.</p></div

    Intron retention assay.

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    <p>Intron retention qRT-PCR results for a subset of genes present in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099493#pone-0099493-t002" target="_blank">Table 2</a>. Intron values for the indicated genes are normalized against the nearest exons and the βactin mRNA values. Data are shown as relative to HeLa values (blue dotted line). Each analysis is an average of at least three independent experiments. The error bars shown are the SEM, ** p value <0.005, * p value <0.05.</p

    List of proteins identified as co-enriched with REIIBP by mass spectrometry.

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    <p>Proteins are ordered relative to the number of mass spectra assigned to peptide sequences unique to each protein. Only proteins with at least 10 fold enrichment in spectral counts relative to control are shown. Sequence coverage denotes the percentage of total amino acid sequence covered by the peptides detected. Asterisks denote known members of the SMN complex: GEMIN4, GEMIN5, GEMIN3 (ddx20), SMN and MEP50 (wdr77).</p

    REIIBP SET domain controls spliceosomal snRNPs abundance.

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    <p>A) Cellular snRNPs abundance. snRNPs were immunoprecipitated with anti SmB/B′ antibody, snRNAs were purified, reverse transcribed and quantified by qPCR, (compared to the level of βactin mRNA present in the input). Each analysis is an average of three independent IPs. B) SMN complex components and SmB/B′ protein levels. Total cell lysates were immunoblotted against the relative antibodies. The black arrow indicates GEMIN3 specific bands. c) snRNAs expression analysis. qRT-PCR of total RNA. Each value is an average of three independent analysis. Figures A and C are expressed relative to control (HeLa cells, dotted line). The error bars are the SEM. ** p value <0.006, * p value <0.05.</p
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