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. 2022 Dec 5;54(12):1881–1894. doi: 10.1038/s41588-022-01236-3

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a, UMAP of all fresh tumor cells, highlighting identified clusters. b, Marker genes (y axis) of identified fresh tumor cell clusters, grouped and annotated on the x axis. Dot sizes represent the percentage of cells expressing the gene in the given cluster, and the color scale shows scaled average relative expression. c, Heatmap representing the relative expression (color bar) of the top 30 marker genes (rows) for the tumor metaprograms identified by NMF across all fresh tumor cells (columns). d, Proportions (y axis) of fresh tumor-derived NMF metaprograms (color legend) in tumor cells for each fresh sample (x axis). e, Cell type-specific TF regulatory networks (regulon, x axis) derived by SCENIC, plotted against their normalized specificity score (y axis). f, Boxplots representing relative frequencies of metaprograms in all fresh and frozen tumors in adult (n = 10) versus pediatric (n = 23) age groups. The median is marked by the thick line within the boxplot, the first and third quartiles by the upper and lower limits, and the 1.5 times interquartile range by the whiskers. Three asterisks denote credible statistical changes as assessed by a Bayesian scCODA model with FDR < 0.05 and without multiple test corrections. g, Boxplots representing relative frequencies of metaprograms in all fresh and frozen tumors grouped by pontine (n = 19) versus thalamic (n = 14) locations. The median is marked by the thick line within the boxplot, the first and third quartiles by the upper and lower limits, and the 1.5 times interquartile range by the whiskers. Three asterisks denote credible statistical changes as assessed by a Bayesian scCODA model with FDR < 0.05 and without multiple test corrections. h, RNA in situ hybridization for MES-like (CD44) and macrophage (CD14) markers in two adult and two pediatric H3-K27M DMGs. Two to three slides were stained for each sample with 10–15 fields of view taken per slide. i, Two-dimensional representations of the OC-like versus AC-like (x axis) and OPC-like (y axis) scores for adult and pediatric H3-K27M DMGs, respectively.