Fig 2: MYCN Drives Transformation of NES Cells to Medulloblastoma(A) Schematic showing differentiation of iPSCs to NES cells, transduction with FLAG-MYCN, and orthotopic implantation into mice.(B) Western blot showing misexpressed MYCN in normal WTC10 NES cells.(C) Empty vector and MYCN NES cells were treated with 5-ethynyl-2'-deoxyuridine (EdU) for 1 h and analyzed via flow cytometry. MYCN NES cells show increased EdU incorporation and S-phase fraction. Data are presented as mean ± SEM; *p < 0.05 (t test).(D) CyQuant Direct cell proliferation analysis showing increased proliferation in MYCN NES cells. Data are presented as mean ± SEM; *p < 0.05 (t test).(E) Kaplan-Meier survival curve of mice injected with empty vector and MYCN NES cells (n = 4). p = 0.004 (log-rank test).(F) (Left) Low magnification (50×) of H&E staining of WTC10 MYCN tumors showing implanted MYCN NES cells expanding and distorting the mouse cerebellum and invading down Virchow-Robin spaces (arrow). (Middle) High magnification (400×) of H&E staining revealing anaplastic features including frequent mitoses (arrows), cell-cell wrapping (black arrowhead), prominent nuclei (red arrowhead), and karyorrhexis (red arrow) characteristic of large cell and/or anaplastic medulloblastoma. (Right) Immunohistochemical staining for synaptophysin (SYP) highlighting neuroblastic pseudorosettes. Scale bars represent 200 µm for 50× images and 20 µm for 400× images.See also Figures S1 and S2 and Tables S1, S4, and S6.

Fig 3: RAD51 Expression Is Elevated in Patient-Derived Glioma Cells(A and B) Representative images of immunofluorescence (IF) staining for RAD51 in three GSCs in comparison with normal human astrocytes (NHAs) (A), quantified in (B) (n = 6 independent experiments with =100 cells counted per cell line).(C) Western blots probed for RAD51 or ß-actin in three GSCs and NHAs.(D and E) Distributions of RAD51 and SOX2 expression (mRNA levels) in single GBM1 cells (n = 273 cells). The dotted line in (E) delineates cells with low and high SOX2 expression.(F) RAD51 expression levels in SOX2-low (n = 30 cells) and SOX2-high (n = 243 cells) populations.(G) Representative immunohistochemistry images from patient tumor samples stained for NES (brown) and RAD51 (red) (left hand panels) or SOX2 (brown) and RAD51 (red) (right hand panels). Examples of cells positive for both proteins are indicated with black arrows, cells negative for both with white arrows, cells positive for just NES or RAD51 indicated by open arrows. Statistical significance calculated by Student’s t test.(H) Quantification of RAD51 and stem marker co-expression from ten tumor samples.Error bars indicate SEM. Statistical significance, unless otherwise stated, calculated by one-way ANOVA. ***p=0.001. Scale bars, 100 µm.

Fig 4: Knockout of GSE1, but not KDM3B, in Gorlin 1 NES Cells Accelerates Tumorigenesis(A) Western blot showing loss of expression of GSE1 (top) and KDM3B (bottom) in response to CRISPR/Cas9 knockout.(B) EdU assay of Gorlin 1 NES cells with control (Ctrl), GSE1, or KDM3B sgRNA. Data are presented as mean ± SEM.(C) CyQuant Direct cell proliferation assay of Gorlin 1 NES cells with Ctrl, GSE1 or KDM3B sgRNA. Data are presented as mean ± SEM.(D) Kaplan-Meier curve showing reduced survival of mice injected with Gorlin 1 NES cells harboring GSE1 sgRNA compared with Ctrl sgRNA or KDM3B sgRNA (n = 5). p = 0.0032 (log-rank test).(E) PCA (left) and SVM (right) classification showing three GSE1-/- tumors subgrouped with SHH medulloblastoma.(F and G) GSE1-/- cell lines were transduced with empty vector or the silent mutant GSE1 (GSE1WT). Both cell lines were (F) analyzed by western blot analysis and (G) injected into mice (n = 5). p = 0.0031 (log-rank test).See also Figures S5 and S6 and Table S3.

Fig 5: Overview of samples underwent subsequent resections. Frequency of cells with different stainings for tissues: (A)—DCX+ cells, (B)—NES+ cells, (C)—OLIG2+ cells. Frequency of cells with mixtures of all stainings for tissues: (D)—OLIG2+DCX+ cells, (E)—OLIG2+NES+ cells, (F)—NES+DCX+ cells, (G)—DCX+OLIG2+NES+ cells.