Figure 6: Haematopoietic ANGPTL4 deficiency promotes CMP expansion.

(a) Dot plots showing the gating scheme of MP and LSK cells (upper panel), contour plots showing the gating schemes of GMP, CMP and MEP cells (lower panel) in the BM of Ldlr^−/− chimeras with WT or Angptl4^−/− BM on WD for 12 weeks. Right panel shows the quantification these progenitor populations (n=5 per group). Data are mean±s.e.m. *P<0.05 by comparison with data from Ldlr^−/− chimeras with WT BM by unpaired t-test. (b) Number of colonies from the BM isolated from WT and Angptl4^−/− mice using CFU assay. Cells were plated in methylcellulose media for 1 week; colonies were counted (b), resuspended and plated for 1 more week (c) (n=6 per group). Number of colony positive wells from LSKs (d) and CMPs (e) sorted from the BM and plated for 10 days individually in 96 well plates cells in methylcellulose media (n=4 per group; a pool of BM was used for sorting from each group). (f) Number of colonies from 100 μl blood from WT and Angptl4^−/− mice plated in methylcellulose media for 10 days (n=3 per group). (g) Proportion of Ki67-positive proliferating cells from indicated cell type from WT and Angptl4^−/− BM. (h) Cell cycle analysis of GMPs and CMPs from WT and Angptl4^−/− BM showing fraction of cells in G1 and S/G2 phase as determined by DAPI staining (n=5 per group). Cell cycle phase gates were drawn as approximations of the Watson (pragmatic) cell cycle modelling algorithm. (i) Proportion of annexin V positive apoptotic cells from indicated cell types from WT and Angptl4^−/− BM (n=5 per group). (j) Representative fluorescent images and flow cytometry quantification of lipid raft content in CMPs of WT and Angptl4^−/− BM assessed by CTxB staining (n=3 per group). Scale bars, 5 μm. All data are the mean±s.e.m. *P<0.05 by comparison with data from WT BM (b,c,h,i,j) by unpaired t-test.