Figure 1.
Morphology and medullosphere count derived from adherent MB tumor cells.
(A) Representative morphology of adherent cells and medullospheres in P1 and P4. (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.
Figure 2.
Transmission electron microscopy.
MBS from DAOY (A), UW228 (B) and ONS-76 (C) 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) and vacuoles (A). MB cell lines feature a low N:C ratio (D, E): 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 (F) as seen in the high N:C ratio with a kidney-shaped nucleus rimmed by a thin layer of cytoplasm.
Table 1.
Phenotypic analysis of stemness marker expression in medulloblastoma cell lines and in correspondent spheres.
Figure 3.
Analysis of MB and MDB expression by Flow cytometry.
FACS analyses shows expression of progenitor markers, CD133 (A) beta- catenin (B) and nestin (C), 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.
Figure 4.
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).
Figure 5.
Representative image from three independent experiments of Coomassie-stained 2-DE patterns of DAOY(A), UW228 (B) and ONS-76 (C) 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 blue –proteins identified in all cell lines and listed in Table 2; in black-proteins identified in adherent cells and spheres of each cell lines and in red-proteins only identified in spheres. Corresponding identifications are reported in Table 3, 4 and 5.
Table 2.
Proteins identified by MALDI-TOF in all (DAOY, UW228, ONS-76) Adherent and Sphere cell lines.
Table 3.
Proteins identified by MALDI-TOF only in DAOY Adherent and Sphere cell lines.
Table 4.
Proteins identified by MALDI-TOF only in UW228 Adherent and Sphere cell lines.
Table 5.
Proteins identified by MALDI-TOF only in ONS-76 Adherent and Sphere cell lines.
Figure 6.
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.
Figure 7.
The networks identified connect all proteins (nodes) already studied in Stem Cell in the literature. The node size is proportional to the number of papers where the proteins have been studied, while the edge width is proportional to the document number where two proteins have been described together. In the network representation, the group of nodes, white colored, identify the proteins commonly detected in Adh/Sp of the cells analyzed: DAOY (A), UW228 (B) and ONS-76 (C). The proteins overexpressed only in the Sp of each cell types are yellow colored. The specific cell type proteins identified in both Adh/Sp are green colored for DAOY, blue for UW228 and pink for ONS-76.
Table 6.
Biological Process Analysis MB cells.
Figure 8.
The physical interactions network.
Protein-protein interaction are identify by pink edges. The black nodes, white edged represent the physical interactions of the common proteins differentially expressed in all DAOY, UW228 and ONS-76 adherent and sphere cell lines, while Sp overexpressed proteins are yellow edged and specific cell type DAOY(A), UW228 (B) and ONS-76 (C) Sp/Ads are green, blue and pink edged respectively. Grey proteins represented new interactions identified in Genemania proteomic databases. The edge width correspond to the weight of interaction.