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. Author manuscript; available in PMC: 2013 Oct 1.
Published in final edited form as: Exp Hematol. 2012 Jun 19;40(10):837–846. doi: 10.1016/j.exphem.2012.06.007

Fig. 5. Scl rescues defective primitive erythroid development in Eng−/− ES cells.

Fig. 5

(A) Eng−/− ES cells infected with an inducible lentiviral vector encoding Scl-iresGFP were selected following three rounds of sorting for GFP. Representative FACS plots show (A) sorting gate for GFP (high expressers) at the second round purification of iScl:Eng−/− ES cells, and (B) induction of Scl expression, as indicated by GFP expression, in day 3.25 iScl:Eng−/−EBs following dox induction from day 2 of EB differentiation. Solid line represents levels of GFP in Scl-induced EBs, whereas dashed line denotes non-induced control (no dox). (C) Western blot confirms induction of Scl in dox-induced iScl:Eng−/− day 3.25 cultures. GAPDH was used as loading control. (D) At day 4.25 of EB differentiation, iScl:Eng−/− ES cells were assayed for primitive erythroid activity. Dox was added to the EB cultures from day 2 of EB differentiation. In one experimental arm, dox was also added to the hematopoietic medium (black bar). Error bars indicate standard errors from 3 independent experiments. *p<0.05, **p<0.01. (E) A representative FACS profile for c-Kit and CD41 expression in non-induced and SCL-induced Eng−/− day 6 EBs, and respective graphic representation on the frequency of c-Kit+CD41+ cells. Error bars indicate standard errors from 4 independent experiments. ***p<0.001. Dox was added from day 2 to day 6 of EB differentiation. (F) Relative levels of Flk-1, Scl, Lmo2, Runx1, Gata1, Gata2, embryonic and adult globins in iScl:Eng−/− EBs at days 3.25, 4.25, and 6 of differentiation. Transcripts are normalized to control non-dox group. Error bars indicate standard errors from 2 independent experiments performed in triplicate.*p<0.05,** p<0.01, *** p<0.001.