Supervised hierarchical clustering analysis of our CNS-PNET model tumors (transcription factors and receptors).

<p>List of the 225 transcription factors and receptors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173106#pone.0173106.s004" target="_blank">S1 Table</a>), which are consistently expressed in our tumors at intermediate or high level of expression (similar or higher than average house-keeping gene expression level).</p

BTIC markers in our CNS-PNET model tumors.

<p>A: Ki67, 20X, insert- 5X digital (LCAS-R-12 weeks post-injection) B: Oct3/4, 40x, insert- 5X digital (LCAS-R-12 weeks post-injection) C: Nestin 20x, insert- 5X digital (LCAS-R-12 weeks post-injection) D: Nestin 40x, insert- 5X digital (LCAS-R-12 weeks post-injection) E: Sox2 40X, insert- 5X digital, (LC26-R-12 weeks post-injection) F: Sox2 40X, insert- 5X digital (LCAS-R-12 weeks post-injection) G: Vimentin 5X, insert- 5X digital (LCAS-R-12 weeks post-injection) H: Vimentin 20X, insert- 5X digital (LC26-R-12 weeks post-injection) TU-tumor; P-parenchyma; V-ventricle; CP-choroid plexus.</p

cMYC, c-MYC-(Phospho S62), HIF-1α, HIF-2α in our CNS-PNET model tumors.

<p>A: cMYC 40X, insert- 5X digital (LCAS-R-12 weeks post-injection) B: c-MYC-(Phospho S62) 20X, insert- 5X digital (LCAS-R-12 weeks post-injection) C: MAX 40X, insert- 5X digital (LCAS-R-12 weeks post-injection) D: MAX 20X (LCAS-R-12 weeks post-injection) E: HIF-1α 40X, insert- 5X digital (LCAS-R-12 weeks post-injection) F: HIF-1α 20X, insert- 5X digital (LCAS-R-12 weeks post-injection) G: HIF-2α 5X, insert- 5X digital (LCAS-R-12 weeks post-injection) H: HIF-2α 40X, insert- 5X digital (LCAS-R-12 weeks post-injection) TU-tumor; P-parenchyma; V-ventricle; N-necrosis.</p

Western blot.

<p>Protein quantitative difference between the RG cells grown for 24 days in normoxic versus hypoxic conditions. The quantitative differences of the proteins calculated after the correction for actual protein loaded per lane using the GAPDH protein control.</p

Supervised hierarchical clustering analysis of our CNS-PNET model tumors (molecular markers).

<p>List of molecular markers of CNS -PNET according to Picard’s classification. Gene symbol_1—primitive-neural (group 1) marker, Gene symbol_2—oligo-neural (group 2) marker, Gene sybmol_3—mesenchymal (group 3) marker.</p

Expression of TP53, YB1 and MDM2 in our CNS-PNET model tumors

<p>A. TP53 40x, insert- 5X digital (LCAS-R) B: TP53 40x, insert- 5X digital (LCAS-R-12 weeks post-injection) C: TP53 40x, insert- 5X digital (LC26-R-12 weeks post-injection) D: YB-1 40X, insert- 5X digital (LCAS-R-12 weeks post-injection) E: MDM2 5x, F: MDM2 40x, (LCAS-R-12 weeks post-injection) TU-tumor; P-parenchyma; V-ventricle.</p

3D imaging of the mice brain using a confocal microscope at one month A- or two months B- post—injection (LCAS-R).

<p>Top left panels reconstitute images of the brains placed horizontally, top right panels reconstitute coronal sections at the indicated lines 1–3. Bottom left panels reconstitute 3D image of the brains oriented as shown by the red plot lines. The section images at the white dotted lines shown at the bottom right panels. A—anterior; P—posterior; R-right; L—left.</p

GFP and Ki-67 expression do not overlap in the majority of the cells in cell lines or in the tumors derived from them.

<p>A- LC25-R-p9, GFP (1:750), 40X; B- LC25-R-p9, Ki-67(1:200), 40X; C- LCAS-R, GFP (1:750), 5X (insert 40x digital); D- LCAS-R, Ki-67 (1:200), 5X (insert 40x digital).</p

Western blot.

<p>TJP1 protein quantitative difference between the RG cell lines grown for 72 hours in normoxic versus hypoxic conditions. The quantitative differences of TJP1 protein calculated after the correction for actual protein loaded per lane using the β-Actin protein control.</p

Ossification within tumor mass (LCAS-R—12 weeks post-injection).

<p>A, B, C. Mature and newly formed bone is observed in intimate correlation with the tumor mass (HE- 10X, 20X and 40X respectively). D, E and F. Bone marrow is observed within mature bone. It shows hematopoietic cells including erythrocytes, leucocytes and megakaryocytes in different stages of maturation suggesting active hematopoietic activity (HE- 10X, 20X and 40X respectively). BM- bone marrow, TU-tumor.</p