297692.fig.001a
(a)
297692.fig.001b
(b)
297692.fig.001c
(c)
297692.fig.001d
(d)
297692.fig.001e
(e)
297692.fig.001f
(f)
297692.fig.001g
(g)
297692.fig.001h
(h)
297692.fig.001i
(i)
Figure 1: Definition of the minimal NHR2 sequence required for RUNX1/ETO inhibitory function. (a) Lentiviral vector SiEW and tested NHR2-containing constructs. (b) Overexpression of the individual proteins in 293T cells and detection of the Flag-tagged constructs by Western blotting. The marker protein eGFP serves as a loading control. (c) Binding of all NHR2-containing proteins to ETO. Co-transfection of 293T cells with the respective constructs together with a plasmid coding for the ETO protein. Immunoprecipitation of the individual protein complexes was performed with an anti-Flag antibody. (d–f) Cellular effects following lentiviral expression of the different NHR2 constructs in Kasumi-1 cells. Time course of the percentage of eGFP-positive cells (d), growth curve of the transduced cells (e), and time course of the expression of the progenitor cell marker CD34 for transduced Kasumi-1 cells (f). (g) Scheme of N68 based NHR2 deletion forms. Indication of the 7 alpha-helical loops L1-L7 of the NHR2 domain. (h) Comparison of N89 and codon-optimized N89 expression levels by western blotting. (i) Percentage of transduced cells in cocultures expressing N89 and the codon-optimized version thereof.