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. 2024 Aug 23;38(10):2171–2182. doi: 10.1038/s41375-024-02373-w

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 5 — A Experimental workflow of genomic/transcriptomic-coupled characterization of single-cell derived colonies by means of ddPCR. Evaluation of CD274 (B, C), POU5F1 (D, E), and NANOG (F, G) expression at the transcriptional level in single-cell derived colonies by ddPCR. In (B, D, and F) colonies (n = 54) were split according to the number of JAK2 mutated alleles and 9p ploidy status. The light blue area highlights +9p colonies. On the other hand, colonies were split according to 9p ploidy status in (C, E, and G). Results are represented as mean + SEM. H–J POU5F1 and NANOG silencing in CD34+ cells from HOM (n = 4) and +9p (n = 6) patients. H Ratio between the total number of colonies grown in methylcellulose-based clonogenic assays in OCT4siRNA+NANOGsiRNA and NTsiRNA samples, split between HOM and +9p patients. Results are represented as mean + SEM. I Ratio between the number of colonies grown in methylcellulose-based clonogenic assays in OCT4siRNA + NANOGsiRNA and NTsiRNA samples, split according to the different Colony-Forming Units (CFUs) in HOM and +9p patients. Results are represented as mean + SEM. J Barplot depicting the frequency of diploid and +9p colonies in +9p patients (n = 6) after POU5F1 and NANOG silencing or NTsiRNA nucleofection. RQ: relative quantity; 1 mut: one JAK2V617F-mutated allele; 2 mut: two JAK2V617F-mutated alleles; 2n: diploid colonies; 3n: +9p colonies; CFU-E/BFU-E: Colony-Forming Unit—Erythroid/Burst-Forming Unit—Erythroid; CFU-GM: Colony-Forming Unit—Granulocyte/Macrophage; CFU-G = Colony-Forming Unit—Granulocyte; CFU-M = Colony-Forming Unit—Macrophage; CFU-GEMM = Colony-Forming Unit—Granulocyte/Erythrocyte/Macrophage/Megakaryocyte; HOM: JAK2-mutated homozygous patients; +9p: JAK2-mutated patients with 9p trisomy; OCT4 + NANOG: POU5F1 siRNA + NANOG siRNA; NT siRNA: Non Targeting siRNA. *p < 0.05; **p < 0.01. A Was created with Biorender.com.