Figure 7: Hoxa9 expression level is critical for transformation from MDS to AML.

(a) Quantitative RT–PCR analysis of the expression of HOXA9 in CD34+ cells of seven MDS–RCMD, seven MDS–RAEBI/II, five MDS/AML, four de novo AML patients, four MLL-AF9-positive AML cell lines (THP-1, NOMO-1, MOLM-13 and MONO-MAC1) and six healthy controls. Scale bars and asterisks show mean±s.d., **P<0.01, and ***P<0.001 by Mann–Whitney U-test. (b,c) Clonogenic capacity of Rx291/Ezh2Δ/Δcells overexpressing Hoxa9. WT and Rx291/Ezh2Δ/ΔHSCs were transduced with a Hoxa9 retrovirus and plated in methylcellulose media, then replated into the same medium every 14 days. The data are shown as mean±s.e.m. (n=3) in b. Expression of exogenous Flag-tagged Hoxa9 protein in colony cells was detected by western blotting using an anti-Flag antibody in c. β-Actin was detected as a loading control. (d) Morphology of AML cells from BM of recipients infused with Hoxa9-expressing Rx291/Ezh2Δ/Δ cells observed by May–Grüenwald Giemsa staining. Scale bar, 20 μm. (e) Fluorescence-activated cell sorting profiles of BM cells from mouse in d and Rx291/Ezh2Δ/Δ-MDS mouse. (f) A proposed model of the role of Ezh2 loss in the development of RUNX1S291fs-induced MDS.