XAV939 affects the DNA repair efficacy of MB cell lines.

<p>Neutral Comet Assay was performed on IR treated cells (10 Gy), with or without XAV939 administration, at different time points post radiations (0, 16, and 24 h). Histograms represent mean ± s.e. of TM measured in at least three independent experiments for each treatment and time point. The greatest TM was observed immediately after IR treatment in both cell lines (A: DAOY, B: ONS-76). XAV939 induces enhanced TM values at 16 h and 24 h after IR compared to DMSO irradiated cells indicating a minor DNA repair capacity. On the right of the figure, a representative example of comets for each treatment obtained in DAOY (upper) and ONS-76 (down) cells.</p

XAV939 inhibits TNKS PARP-activity in MB cell lines.

<p>WB analysis and densitometry of total and nuclear MB cells extracts after XAV939 treatment: MB cell lines (DAOY, ONS-76) were treated with 5 μM XAV939 or with an equal volume of DMSO. <b>A.</b> Both cell lines showed an increase in total Axin protein levels at 8 h after treatment (80% and 70% respectively in ONS-76 and DAOY compared to DMSO treated control, <i>p</i> <. 05), followed by a β-catenin decrease in total (30%, <i>p</i> <. 05) and, in particular, in nuclear extracts, at 16 h after drug administration (80% reduction compared with control, <i>p</i> <. 001). <b>B.</b> XAV939 induced a DNA-PKcs protein level reduction of about 40% at 8 h and 16 h after treatment compared to non-treated control cells (<i>p</i> <. 05 in A, <i>p</i> <. 01 in B). Densitometry data (mean ± s.e.) were normalized with β-actin (for total extracts) and lamin-b (for nuclear extracts) and are representative of the results derived from three independent experiments.</p

XAV939 impairs the clonogenic and proliferative capacity and enhances MB cells radio-sensitivity.

<p><b>A.</b><i>Growth curves assay</i>. XAV939 (5 μM) treated cells and IR (2 Gy) treated cells showed a similar growth rate with about 40% (DAOY) and 30% (ONS-76) reductions compared to DMSO control cells (<i>p</i> <. 01). In both cell lines, the co-administration of XAV939 and IR induces a massive cell proliferation inhibition with a decrease of about 70% (<i>p</i> <. 001). Each point represents the mean ± s.e. of three independent assays. <b>B.</b><i>Clonogenic forming assay</i>. XAV939 alone (5 μM) inhibits a clone-forming ability in both MB cell lines as well as IR treatment (2 Gy), with a clonogenic capability reduction of about 38% (<i>p</i> <. 01). The IR and drug co-administration induces a drastic inhibition of clone-forming in both cell lines (70% reduction in DAOY cells and 81% in ONS-76 compared with control cells, <i>p</i> <. 001).</p

Biological Process Analysis MB cells.

<p>Biological Process Analysis MB cells.</p

Transmission electron microscopy.

<p>MBS from DAOY (<b>A</b>), UW228 (<b>B</b>) and ONS-76 (<b>C</b>) show a uniform stem-like aspect characterized by a high N:C ratio; nucleus is irregularly shaped and cytoplasm appears sparsely organized with unspecific electron-dense organelles (<b>B</b>) and vacuoles (<b>A</b>). MB cell lines feature a low N:C ratio (<b>D, E</b>): nuclei exhibit an irregular profile and are peripherally located; the cytoplasm is populated by many organelles, such as RER, Golgi apparatus and mitochondria. On the contrary, ONS MB seem to maintain stemness appearance (<b>F</b>) as seen in the high N:C ratio with a kidney-shaped nucleus rimmed by a thin layer of cytoplasm.</p

Proteins identified by MALDI-TOF only in ONS-76 Adherent and Sphere cell lines.

a<p>Abb, abbreviation.</p>b<p>AC, accession number.</p>c<p>MW, molecular weight.</p>d<p>pI, isoelectric point.</p>e<p>NVM, number of matched mass values on number of total mass values searched.</p>f<p>C%, the sequence coverage, which is calculated as the percentage of identified sequence to the complete sequence of the matched protein.</p>g<p>MS, mascot score.</p>h<p>CC, culture condition.</p

Analysis of MB and MDB expression by Flow cytometry.

<p>FACS analyses shows expression of progenitor markers, CD133 (<b>A</b>) beta- catenin (<b>B</b>) and nestin (<b>C</b>)<b>,</b> evaluated in both MB and in MBS for all cell lines by flow cytometry. Graphs represent pooled data from three independent determinations. Standard deviations and Student’s T-test were calculate to compare MB and MBS. Probability values less than 0.01 were considered significant.</p

Immunostaining of TMA.

<p>Staining for Oct-4, Sox-2, beta Catenin and Nucleophosim on DAOY, ONS76 and UW228 cell lines grown as spheres are shown at 40x magnification. UW228 (second column) show smaller spheres and less intense staining for Oct-4 and Sox-2.</p

Proteomic analysis.

<p>Representative image from three independent experiments of Coomassie-stained 2-DE patterns of DAOY(<b>A</b>), UW228 (<b>B</b>) and ONS-76 (<b>C</b>) cell lines. Proteins were separated using 7-cm, pH-3-10 NL strips followed by SDSPAGE on 10%, 7×10-cm gels. Proteins showing differential expression were indicated with different colors: in <i>blue</i> –proteins identified in all cell lines and listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063748#pone-0063748-t002" target="_blank">Table 2</a>; in <i>black</i>-proteins identified in adherent cells and spheres of each cell lines and in <i>red</i>-proteins only identified in spheres. Corresponding identifications are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063748#pone-0063748-t003" target="_blank">Table 3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063748#pone-0063748-t004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063748#pone-0063748-t005" target="_blank">5</a>.</p

Phenotypic analysis of stemness marker expression in medulloblastoma cell lines and in correspondent spheres.

<p>The table represents mean standard deviation of three independent experiments. Numbers in bold indicate a statistically significant difference of spheres with reference to the control adhesion conditions (*p<0,01).</p