Extended Data Fig. 1: Cell sorting gates and dynamics of IRE1α-XBP1 activity and expression in normal bone marrow hematopoietic stem and progenitor cells.
(a and b) Representative flow cytometry gates used for the identification and sorting of mouse (a) and human (b) hematopoietic stem and progenitor cells (HSPC). Human HSPC populations were defined follows: HSC, Lineage (Lin)-CD34+CD38-CD45RA-CD90+CD49f+; MPP, Lin-CD34+CD38-CD45RA-CD90-CD49f-; MLP, Lin-CD34+CD38-CD90-CD45RA+; CMP, Lin-CD34+CD38+CD10-CD45RA-Flt3+; GMP, Lin-CD34+CD38+CD10-CD45RA+Flt3+; MEP, Lin-CD34+CD38+CD10-CD45RA-Flt3-. Mouse HSPCs were defined as follows: HSC, Lin-c-Kit+Sca1+CD150+CD48-; MPP, Lin-c-Kit+Sca1+CD150-CD48-; HPC, Lin-c-Kit+Sca1+CD150-CD48+; CMP, Lin-c-Kit+Sca1-CD34+CD16/32-; GMP, Lin-c-Kit+Sca1-CD34+CD16/32+; MEP, Lin-c-Kit+Sca1-CD34-CD16/32-. (c-e) Gene expression in mouse HSPC subsets and GMPs determined by qRT-PCR. Expression of Ern1, Xbp1, and Xbp1s is normalized to Actb. Data are representative of three independent experiments. *, P < 0.05; **, P < 0.01; ns, not significant; Students t-Test. (f) Relative (±s.e.m., normalized to HSC) XBP1S/XBP1 ratio in human BM progenitor and mature CD14+ cells sorted from BM mononuclear fraction (N = 6 donors). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant; Students t-Test. (g) Flow cytometry detection of reactive oxygen species (ROS) by dihydroethidium (DHE) staining in total bone marrow (BM) c-Kit+ cells, LSK and GMP cells from Cybb+/+ and Cybb-/- mice. Unshaded histograms represent unstained control sample. Graphs show relative (average ± S.D., N = 4 mice/group) DHE mean fluorescence intensity (MFI). Mice were 3-4 months old mice. *, P < 0.05; **, P < 0.01; ***, P < 0.001; Students t-Test. (h) Unfolded proteins were measured by staining with the thiol probe tetraphenylethene maleimide (TPE-MI). Data show average (± S.D., N = 5 mice/group) TPE-MI MFI in age-matched 3-4 months old mice. *, P < 0.05; ns, not significant; Students t-Test.
