Figure S1.
N-cad expression, depletion, and controls for PHGG migration assays. (A) Overall survival of pediatric brain tumor patients with high or low N-cad mRNA expression. Data collected from Pediatric Brain Tumor Atlas (PBTA) (http://pedcbioportal.kidsfirstdrc.org, PedcBioPortal, Open Pediatric Brain Tumor Atlas, v22). (B–D) Migration or invasion distance of PBT-04 and PBT-05 cells in mouse cerebellar neurons, Matrigel, and laminin/fibronectin. Unpaired t tests. *P < 0.05, ***P < 0.001, ****P < 0.0001. (E) mRNA expression levels of cadherins in patient-derived PHGG tissues and cells (https://r2.amc.nl, R2: Genomics Analysis and Visualization, Mixed Pediatric PDX-Olson). (F) N-cad protein expression level in DMG (PBT-22FH, PBT-24FH, and PBT-29FH) and PHGG (PBT-04FH and PBT-05FH) cell lines and mouse cerebellar neurons and mouse astrocytes. β-Tubulin is shown as a loading control. (G) Western blots for control or N-cad shRNAs in PBT-05 cells. Vinculin is shown as a loading control. (H) RNA levels of N-cad and other cell adhesion proteins in control or N-cad shRNA PBT-05 cells. Mean CPM (counts per million) from four biological replicates. Lines connect paired samples. Adjusted P values from the Wald test using the Benjamini-Hochberg method. (I–K) N-cad does not regulate spheroid formation or cell proliferation. (I) Representative images of spheroids. Scale bar, 100 μm. (J) Spheroid diameter at start of migration experiment. Ordinary one-way ANOVA Holm-Šídák’s multiple comparisons test. ns, not significant. N = 13–14 spheroids, four experiments. (K) EdU pulse labeling. Ordinary one-way ANOVA Holm-Šídák’s multiple comparisons test. ns, not significant. N = 5–7 spheroids, three experiments. (L and M) Western blots and migration distance on laminin of control and N-cad siRNA-treated PBT-05 cells. Unpaired t test. ***P < 0.001. N = 15–19 spheroids, 4 experiments. (N and O) N-cad depletion increases single-cell migration. Representative images of migrating control or N-cad shRNA spheroids on laminin or 3D Matrigel. Scale bars, 200 or 50 μm (inset). (P and Q) Controls for migration on astrocytes and astrocyte ECM. (P) Cell migration on mouse astrocytes. PBT-05 cells were identified with human-specific anti-vimentin antibodies. PBT-05 cell and astrocyte actin were detected with phalloidin. (Q) Decellularized astrocyte cultures lack N-cad. Scale bars, 200 μm. Error bars indicate mean ± SEM. Source data are available for this figure: SourceData FS1.

N-cad expression, depletion, and controls for PHGG migration assays. (A) Overall survival of pediatric brain tumor patients with high or low N-cad mRNA expression. Data collected from Pediatric Brain Tumor Atlas (PBTA) (http://pedcbioportal.kidsfirstdrc.org, PedcBioPortal, Open Pediatric Brain Tumor Atlas, v22). (B–D) Migration or invasion distance of PBT-04 and PBT-05 cells in mouse cerebellar neurons, Matrigel, and laminin/fibronectin. Unpaired t tests. *P < 0.05, ***P < 0.001, ****P < 0.0001. (E) mRNA expression levels of cadherins in patient-derived PHGG tissues and cells (https://r2.amc.nl, R2: Genomics Analysis and Visualization, Mixed Pediatric PDX-Olson). (F) N-cad protein expression level in DMG (PBT-22FH, PBT-24FH, and PBT-29FH) and PHGG (PBT-04FH and PBT-05FH) cell lines and mouse cerebellar neurons and mouse astrocytes. β-Tubulin is shown as a loading control. (G) Western blots for control or N-cad shRNAs in PBT-05 cells. Vinculin is shown as a loading control. (H) RNA levels of N-cad and other cell adhesion proteins in control or N-cad shRNA PBT-05 cells. Mean CPM (counts per million) from four biological replicates. Lines connect paired samples. Adjusted P values from the Wald test using the Benjamini-Hochberg method. (I–K) N-cad does not regulate spheroid formation or cell proliferation. (I) Representative images of spheroids. Scale bar, 100 μm. (J) Spheroid diameter at start of migration experiment. Ordinary one-way ANOVA Holm-Šídák’s multiple comparisons test. ns, not significant. N = 13–14 spheroids, four experiments. (K) EdU pulse labeling. Ordinary one-way ANOVA Holm-Šídák’s multiple comparisons test. ns, not significant. N = 5–7 spheroids, three experiments. (L and M) Western blots and migration distance on laminin of control and N-cad siRNA-treated PBT-05 cells. Unpaired t test. ***P < 0.001. N = 15–19 spheroids, 4 experiments. (N and O) N-cad depletion increases single-cell migration. Representative images of migrating control or N-cad shRNA spheroids on laminin or 3D Matrigel. Scale bars, 200 or 50 μm (inset). (P and Q) Controls for migration on astrocytes and astrocyte ECM. (P) Cell migration on mouse astrocytes. PBT-05 cells were identified with human-specific anti-vimentin antibodies. PBT-05 cell and astrocyte actin were detected with phalloidin. (Q) Decellularized astrocyte cultures lack N-cad. Scale bars, 200 μm. Error bars indicate mean ± SEM. Source data are available for this figure: SourceData FS1.

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