Journal: Nature Communications
Article Title: MAEA is an E3 ubiquitin ligase promoting autophagy and maintenance of haematopoietic stem cells
doi: 10.1038/s41467-021-22749-1
Figure Lengend Snippet: 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: NH 4 Cl. 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.
Article Snippet: To measure the half-life of surface receptors, BM cells were incubated in StemSpan SFEM (Stem Cell Technology #09650) with 50 μM cycloheximide (Millipore Sigma #508739) at 37 °C.
Techniques: Electron Microscopy, Immunofluorescence, Isolation, Cell Culture, Ex Vivo, Incubation, Irradiation, Transplantation Assay
