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S.G. Kellaway et al.
induced RUNX1-EVI1 expression in newly forming HP (Online Supplementary Figure S1B), when Runx1 becomes upregulated as shown in the schematic in Figure 1A. These time points were used for all subsequent experi- ments, with the cells sorted from the day 2 blast culture being used for genome-wide analysis. Since differentia- tion in this system is transient, we also used cell sorting to obtain cells from earlier differentiation stages which enabled us to study the effect of RUNX1-EVI1 expression at these stages as well. We titrated induction of RUNX1- EVI1 such that the protein expression was at a level simi- lar to that seen in the SKH1 cells, and gene expression was at a similar level to endogenous RUNX1, which itself was
stably expressed (Figures 1B and C).
Induction of RUNX1-EVI1 led to a partial disruption of
the EHT following 18 hours of dox induction (Figure 1D), with a reduction in cKit+ Tie2- CD41+ HP and a reciprocal increase in hematopoietic committed HE cells (HE2, cKit+ Tie2+ CD41+). This result is concordant with the notion that RUNX1-EVI1 acts as a dominant negative to RUNX1, since the earlier uncommitted endothelial cells which had not yet upregulated Runx1 (HE1, cKit+ Tie2+ CD41-) were unaffected. Inducing RUNX1-EVI1 prior to the EHT ham- pers hematopoietic differentiation leading to a consider- ably greater proportion of Tie2- CD41- negative cells (Online Supplementary Figure S1C).
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C
E
Figure 2. RUNX1-EVI1 expression causes reduced cell cycling and colony forming capacity in hematopoietic progenitors. (A) Fewer floating hematopoietic progenitor (HC) cells were present in day 2 blast culture, following induction of RUNX1-EVI1, n=5, **P<0.01. Cell cycle stages, n=3. (B) and quiescence, n=5 (C) were assessed in the whole blast culture at the same time-point, showing an increased proportion of cells in G0 and G1. Example flow cytometry plots are shown to the right, *P< 0.05, **P<0.01. (D) Floating progenitor cells with RUNX1-EVI1 induced formed fewer colonies, n=3, **P<0.01 (E) Colonies following doxycycline (dox) induction in blast culture only were comprised of approximately equivalent proportions of granulocyte/macrophage (GM), erythroid (Ery) or mixed colonies, with a slight increase in the mixed-type at the expense of singular lineage, n=3. All error bars (A to E) represent standard error of the mean. (F) Representative brightfield images of colonies with and without dox induction in blast culture only.
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