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. 2021 May 4;12:2522. doi: 10.1038/s41467-021-22749-1

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© The Author(s) 2021

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. 4 — a Experimental scheme and evaluation of autophagy flux in control (Ctrl) and Maea^Csf1r-Cre HSCs (n = 7). % autophagy flux is calculated as 100x(1 − (−L/N))/(+L/N). N: NH₄Cl. L: leupeptin. p = 0.0036. b Representative electron microscopy micrographs of control and Maea^Csf1r-Cre HSCs for ultrastructural analysis of the autophagic compartments (red arrows point to an autolysosome in control and an autophagosome in Maea^Csf1r-Cre). c Morphometric analysis of control and Maea^Csf1r-Cre HSCs: quantification of the numbers of autophagic vacuoles (AV) and their break down into number (left) and percentage (right) of autophagosomes (APG) and autolysosomes (AUT), per cell area. n = 27 control and 23 Maea^Csf1r-Cre cells analysed. Analysis were applied to cells blindly from two independent experiments. Unblinding was done during data plotting. p value from left to right are p = 0.035, 0.0245, 0.0306. d Frequencies of HSCs in control and Maea^Csf1r-Cre bone marrow (BM) cells before and after 3 h of starvation in culture (n = 6 each). p value from left to right are 0.0093, 0.0016. e Representative immunofluorescence images and quantification showing subcellular colocalization of FLT3 and LC3 in freshly isolated (Ctrl) and starved (cultured ex vivo in StemSpan with no cytokines but in the presence of lysosome inhibitors N/L for 3 h to induce autophagy) HSCs (n = 11 cells analysed). p = 0.0071. Scale bar = 10 μm. f Flt3 half-life in control and Maea^Csf1r-Cre lineage- Sca-1+ c-kit+ progenitor cells (LSKs) cells incubated in the presence of 50 μM cycloheximide and 10 mM lithium chloride (LiCl) (n = 4 animals). p value from left to right are 0.034, 0.0105, 0.08. g Quantification of LSKs and HSCs in control and Maea^Mx1-Cre BM treated with vehicle (Veh), LiCl or verapamil (Verap) 3 weeks after poly I:C induction (Veh: n = 3, LiCl: n = 6, Verap: n = 6). h Peripheral blood donor chimaerism in CD45.1 lethally irradiated wild-type (WT) recipients at indicated time points after competitive BM transplantation (BMT) of equal number of CD45.1 WT competitor BM cells and CD45.2 donor BM cells from indicated groups (n = 5 each group). Data are shown as mean ± sem. ns not significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by unpaired two-sided t-test with 95% confidence level unless otherwise indicated.