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. 2020 Jul 1;21:157. doi: 10.1186/s13059-020-02058-4

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© The Author(s) 2020

Open AccessThis 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 licence, and indicate if changes were made. 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/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 6 — Functional effect of CDK inhibitors during EHT. a Schematic showing inhibitors used in the experiments (left panel) and their effect on cell cycle progression (right panel). Samples were treated for 48 h between EHT D3 and EHT D5 with either 0.1% DMSO (CTRL), 1 μM PD0332991 (CDK4/6i), 10 μM RO3306 (CDK1i), or 4 μM Roscovitine (CDK2/1i). At EHT D5, cells were either a processed for EdU staining and flow cytometry to monitor cell cycle profile or b stained for flow cytometry to monitor the frequency of distinct cell populations generated (P1, mesenchymal cells; P4, endothelial cells; P2 and P5, erythroid progenitors; P3 and P6, HPCs). Results are representative of 3 independent experiments. c At EHT D5, cells were washed to remove the inhibitors and two populations of HPCs (corresponding to P3 and P6 in b) were FACS-sorted and further cultured in a CFU assay to reveal their differentiation potential. Data are mean ± SEM of 3 independent experiments, with statistical significance compared to control as *P < 0.5, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA. Degrees of freedom in b for CDH5− HPCs are DFn = 1.103, DFd = 2.205, F value 11.19, and for CDH5+ HPCs are DFn = 1.046, DFd = 2.092, F value 204.2. Degrees of freedom in c are DFn = 2, DFd = 6; F values for the distinct colonies in CDH5− HPCs are BFU-E = 2.483, CFU-E = 2.771, CFU-GM = 12.15, CFU-G = 3.261, CFU-M = 0.8633, and CFU-GEMM = 17.43; F values for the distinct colonies in CDH5+ HPCs are BFU-E = 12.51, CFU-E = 6.757, CFU-GM = 67.36, CFU-G = 21.71, CFU-M = 19.26, and CFU-GEMM = 30.63