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

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

Morphology and medullosphere count derived from adherent MB tumor cells.

<p>(<b>A</b>) Representative morphology of adherent cells and medullospheres in P1 and P4. (<b>B</b>) MBS count obtained with MB cell lines during different passages (P1–P10). Measurements were done in triplicate and data are presented as mean ± SD.</p

Proteins identified by MALDI-TOF in all (DAOY, UW228, 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

Migration assay.

<p>Invasion capability of MB cells (ONS-76, DAOY and UW228) and their corresponding MBS (ONS-76 S, DAOY S and UW228 S) at 24h from experimental set up using a transwell (Matrigel) migration assay. The results are representative of two different experiments. Bar charts represent the mean of migrating cells evaluated in two independent experiments. *P<0,05 ***P<0,0001 (unpaired, two tailed t-test).</p

Proteins identified by MALDI-TOF only in DAOY 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