Copy number abnormalities analysis of CD138⁺⁺ and CD138^low cells.

<p>Log ratio plot of all chromosomes corresponding to CD138⁺⁺ RPMI-8266 cells (n = 3; left panel) and CD138^low RPMI-8266 cells (n = 3; right panel) on the basis of Cytoscan HD array generated with Nexus.</p

Genes differentially expressed in CD138^low RPMI-8226 <i>versus</i> CD138⁺⁺ RPMI-8226 cells.

<p>Genes differentially expressed in CD138^low RPMI-8226 <i>versus</i> CD138⁺⁺ RPMI-8226 cells.</p

Using Style to Understand Descriptions of Software Architecture

The software architecture of most systems is described informally and diagrammatically. In order for these descriptions to be meaningful at all, figures are understood by interpreting the boxes and lines in specific, conventionalized ways [5]. The imprecision of these interpretations has a number of limitations. In this paper we consider these conventionalized interpretations as architectural styles and provide a formal framework for their uniform definition. In addition to providing a template for precisely defining new architectural styles, this framework allows for the proof that the notational constraints on a style are sufficient to guarantee the meanings of all described systems and provides a unified semantic base through which different stylistic interpretations can be compared

Proliferation of CD138⁺⁺ and CD138^low subpopulations.

<p>(A) Left: Cell percentage of CD138⁺⁺ and CD138^low RPMI-8226 or NCI-H929 cells in G0-G1, S and G2-M phases. The results are expressed as the means ± SEM of at least three independent experiments. Right: Representative DRAQ5 histograms for each indicated population. (B) Left: Relative Ki-67 MFI of CD138⁺⁺ and CD138^low RPMI-8226 and NCI-H929 cells with respect to isotype control. Results are expressed as the means ± SEM of three independent experiments. Right: Representative Ki-67 histograms for each indicated population. Filled histograms (isotype control); open histograms (Ki-67).</p

Tumorigenic potential of CD138⁺⁺ and CD138^low subpopulations.

<p>(A) 3×10⁴ sorted CD138⁺⁺ RPMI-8226 or CD138^low RPMI-8226 cells were subcutaneously injected into CB17-SCID mice to generate primary tumors. Subsequently, 3×10⁶ cells isolated from selected primary tumors were serially transplanted into new CB17-SCID mice to generate secondary tumors. The engraftment efficacy is indicated in each case. (B, C) Tumor growth curves for CB17-SCID mice that developed measurable primary and secondary tumors. Growth curves represent tumor volumes (means ± SEM; n = 4–6) until the time point in which the first mouse in every group is sacrificed.</p

Study of aldehyde dehydrogenase expression and drug sensitivity in CD138⁺⁺ and CD138^low subpopulations.

<p>(A) Single parameter histograms illustrating the expression of ALDH in the presence or absence of the ALDH inhibitor DEAB (diethylaminobenzaldehyde) in CD138⁺⁺ and CD138^low subpopulations of the cell lines RPMI-8226, NCI-H929 and MM1S (B) Sorted CD138⁺⁺ or CD138^low RPMI-8226 cells were incubated in the absence (control) or presence of bortezomib (10 nM), melphalan (10 μM) or doxorubicin (250 nM) for 24 and 48 hours. After the incubation time, cell viability was measured by MTT assay and the percentage of cell viability was calculated considering control as 100%. Results are the means ± SEM of at least three independent experiments.</p

CD138 expression in MM cell lines.

<p>(A) Dot plots showing the percentage of CD138⁺⁺ and CD138^low cells in MM cell lines. (B) Top: summary of the expression of CD19, CD20, CD27, CD38, CD45 and CD56 in CD138⁺⁺ and CD138^low subpopulations in MM cell lines. Code: – (negative); −/+ (heterogeneity); dim (weak positive); + (positive); ++ (high positive). Bottom: representative dot plots corresponding to non-stained RPMI-8226 cells (negative control; light grey) and stained CD138⁺⁺ (black) and CD138^low (dark grey) RPMI-8226 cells. (C) Expression of CD138 by real-time quantitative PCR in CD138⁺⁺ and CD138^low RPMI-8226 subpopulations. Relative values were calculated by the 2^−ΔCt method (ΔCt = Ct_(Gene)−Ct_(GAPDH)). The <i>GAPDH</i> gene was used as a control gene. Results are expressed as the means ± SEM (n = 3). (D–K) Confocal images corresponding to the immunocytochemistry for CD138 (red) in CD138⁺⁺ and CD138^low RPMI-8226 and NCI-H929 cells. Nuclear DNA was stained with DAPI (blue). Magnification of the lens, 63x. Specific “4× zoom” was made in E, G, I, K.</p

Additional file 1: Figure S1. of Preclinical anti-myeloma activity of EDO-S101, a new bendamustine-derived molecule with added HDACi activity, through potent DNA damage induction and impairment of DNA repair

U266, RPMI-8226, and their derivatives, U266-LR7 and RPMI-LR5 partially resistant to melphalan, were incubated with increasing doses of EDO-S101, and cell viability was analyzed by MTT metabolization. Figure S2. EDO-S101 toxicity, on PCs and B lymphocytes derived from bone marrow samples from 3 MM patients, was evaluated after 48 h of incubation by flow cytometry. Figure S3. EDO-S101 dose response (48 h) of different proteins implicated in DNA damage repair in U266 cell line. Figure S4. Dose response (48 h) of different proteins implicated in DNA damage repair and HDAC inhibitory effect after treatment with EDO-S101 of MM1S in the presence or absence of stromal components of the bone marrow microenvironment. MM1S was incubated with EDO-S101 alone, in co-culture with the human stromal cell line hMSC-TERT, and in co-culture with bone marrow mesenchymal stromal cells from a patient with MM (pBMSC). In all cases, the alkylating and the HDACi effect of EDO-S101 were preserved. Figure S5. Different MM cell lines were incubated with 1 and 2.5 μM EDO-S101 for 48 h. After propidium iodide staining, the cell cycle profile was analyzed by flow cytometry. Calculation of percentages of cells at each phase did not consider cells at G0. Figure S6. Bcl-2 family proteins studied by Western blot after treatment of MM1S with the indicated doses of EDO-S101 for 48 h. Figure S7. Toxicity profile of mice bearing a subcutaneus plasmacytoma and treated with the indicated drug. The EDO-S101 group showed a reversible 10–20% loss of body weight. Each point represents the mean ± SD. Figure S8. The combination of EDO-S101 plus bortezomib was also able to improve the effect of single treatments in RPMI-8266, JJN3, and U266 cell lines. Figure S9. Toxicity profile of mice bearing a subcutaneus plasmacytoma and treated with the indicated drugs. The EDO-S101 + Bortezomib group showed a reversible 10–20% loss of body weight. Each point represents the mean ± SD. (PPTX 348 kb