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. 2023 Sep 4;55(9):1531–1541. doi: 10.1038/s41588-023-01480-1

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© The Author(s) 2023

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. 3 — a, Three-dimensional diffusion map of 8,988 Lin^-CD34⁺ cells from TP53-sAML patients (related to Fig. 2a) colored by expression of an HSC signature (Supplementary Table 3). b, Projection of TP53-WT (n = 880) pre-LSCs on HD (left) and MF (right) hematopoietic hierarchy (related to Fig. 2c and Extended Data Fig. 5d). c, Immunophenotype of Lin^-CD34⁺CD38⁻ cells from four representative sAML patients colored by genotype. Lin^-CD34⁺CD38⁻CD90⁺CD45RA⁻ cells (HSCs) were enriched using the sorting strategy outlined in Extended Data Fig. 2a. d, scVelo analysis of differentiation trajectories of Lin^-CD34⁺ cells from one HD (left) and two representative TP53-sAML patients (middle and right). Insets show HSC or pre-LSCs clusters. e–g, Scores of HSC (e), WNT β-catenin signaling (f) and cell-cycle (g) associated transcription in Lin^-CD34⁺CD38⁻ cells from HDs (n = 730 cells), MF (n = 1,106 cells) and pre-LSCs from TP53-sAML patients (n = 880 cells). Boxplots represent the median, first and third quartiles, and whiskers correspond to 1.5 times the interquartile range; the white square indicates the mean for each group. P indicates the Wilcoxon rank sum test P value. h–j, Functional analysis of pre-LSCs. Schematic representation of HSC in vitro assays (h), LTC-IC colony forming unit activity (i) and short-term in vitro liquid culture clonogenicity (j) of HSC from HDs (n = 4), MF (n = 3) and pre-LSCs from TP53-sAML patients (n = 3, samples used (IF0131, IF0391 and GR001) were known to have TP53-WT pre-LSC in the HSC compartment). Violin plot indicates points’ density; dashed lines, median and quartiles, two independent experiments (i); barplot indicates mean ± s.e.m., three independent experiments with 30 colonies plated per experiment (j). P indicates two-tailed t-test P value. k, GSEA analysis of HALLMARK inflammatory pathways in pre-LSCs compared to HDs; positive NES in the heatmap represents significant (FDR q value < 0.25) enrichment in pre-LSCs, values indicate NES for each pathway.

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