Fig 2: (A) Selected genes downregulated upon KDM5A and PHF2 depletion relative to shControl (RNAseq data). (B) Expression levels of same genes as in “A” in EWS/Fli1-depleted cells relative to control (A673-shA1c cells, −/+ Dox × 72 h; qRT-PCR). (C) Expression levels of same genes as in “A” in EWS/Fli1-depleted SK-N-MC cells relative to control (RNAseq data from [7]). (D) MCAM and (E) L1CAM expression levels in control, KDM5A-depleted and PHF2-depleted A673 and SK-ES-1 cells (qRT-PCR, 2 days following initial selection). (F) Top: H3K4me3 levels in control, KDM5A shRNA-depleted (2 days following initial selection), and CPI-455 treated (48 hours) A673 cells. Bottom: MCAM and L1CAM expression levels in control and CPI-455 treated cells (qRT-PCR at 48 hours; mean and standard deviation of expression in A673 and TC32 cells). (G) Kaplan-Meier analysis of Ewing sarcoma patient survival as a function of high versus low L1CAM expression in tumors (data from [46], via R2, Genomics Analysis and Visualization Platform, https://hgserver1.amc.nl). (H) Expression of L1CAM in patient-derived Ewing sarcoma cell lines and hMSC, with tubulin as loading control. (I) Stable shRNA-mediated depletion of L1CAM in A4573 cells inhibits cell migration and invasion (transwell assay without (migration) and with Matrigel (invasion); mean and standard error from 3 independent experiments, each performed in duplicate; ** p < 0.01, * p < 0.05, # p = 0.056, relative to shControl, one-way ANOVA with multiple comparisons).

Fig 3: (A) KDM5A (left) and PHF2 (right) depletion in EWS/Fli1low cells. Immunoblot data with tubulin as loading control. (B) Colony formation in clonogenic assay, shown as images from representative experiment (top), and quantifications from 2 independent experiments, each performed in triplicate (bottom); * p < 0.05, ** p < 0.01, relative to EWS/Fli1low/shControl, one-way ANOVA with multiple comparisons. (C) Transwell invasion by the indicated cells. Data from 2 independent experiments, each performed in triplicate; mean and standard error; data from EWS/Fli1low/shControl cells set to 1; ** p < 0.01, relative to EWS/Fli1low/shControl, one-way ANOVA with multiple comparisons. (D) Fractional expression of L1CAM in PHF2-depleted EWS/Fli1low cells, relative to EWS/Fli1low/shControl cells; mean and standard error of RNA levels from 2 independent experiments, each performed in triplicate.

Fig 4: Characterization of the plasma‐resident neuronal‐derived extracellular vesicles (nEVs). (A) Total protein content, (B) size distribution, (C) correlation curve, and (D) Z‐average size (nm) of nEVs isolated from healthy donors (HDs) and participants with Down syndrome (DS). Error bars represent standard deviation (SD). In (E‐F), morphological study of nEVs. BF‐TEM image of nEVs (E). Plot showing the frequency distribution of the vesicle size histogram (F). Data fit by Gaussian convolution functions (red line). P < .01 statistically significant difference between experimental and theoretical data fitting. Furthermore, identification of nEV membranes evidences the presence of the bilayer membranes (inset in F). In (G), representative Western blot images of total EVs (T, isolated with Exo‐spin Blood kit EX‐02), EV‐depleted plasma (P, supernatant after Exo‐spin Blood kit EX‐02), and nEVs (after immunocapture with L1CAM) isolated from HD (n = 3) and DS (n = 3) and probed for L1CAM, NeuN, Alix, CD81, and APOA1. The two bands detected for L1CAM in nEVs represent the non‐glycosylated (∼≈170 kDa) and the glycosylated form (∼≈220 kDa). In (H), the degree of neuronal enrichment for nEVs compared to total EVs. L1CAM and NeuN and CD81 densitometric values were first normalized for total load and then both L1CAM and NeuN levels were expressed as a ratio with CD81. Data are presented as means ± SD. *P < .05 versus Total EVs (Mann‐Whitney)