Figure 4.

Effects of Reduced DOT1L Activity and Enhanced Sox2 Levels on the Ratio of Nanog+/E-Cadherin+ Colonies

(A) Left: Scheme of experiment for “Fix” samples in (B)–(D). Dox added on d0, fixed on days indicated. Right: Scheme of experiment for “-Dox” in (B)–(D). Dox added on d0, removed on days indicated, and fixed on d14. No significant difference between N+E+ colonies in (B), (C), (D).

(B) Comparison of N+E+ colonies in “Fix” and “-Dox” samples for NSCs. Counts from three independent experiments are stacked. Note the same data are presented in Figures 1C and 3C.

(C) As in (B) but for astrocytes. Note: same data are presented in Figures 1C and S2B.

(D) As in (B) but for MEFs. Note: same data are presented in Figures 1C and S2C.

(E) Counts of N+, E+ and N+E+ colonies from NSC reprogramming cultures on d10 in the presence of DMSO (left) or DOT1L inhibitor SGC0946 (middle). Counts from three independent experiments are stacked. Western blot analysis of H3K79me2 after SGC0946 treatment for 24 hr. No significant difference in the two conditions by t test.

(F) Relative expression of endogenous Oct4, Sox2, Klf4, and c-Myc in NSC, astrocytes, and MEFs. Error bars are SD of three technical replicates from one representative experiment. Inset, SOX2 expression in GFAP+ astrocytes. Scale bar, 50 μm.

(G) Left: Immunofluorescence images of MEFs following infection with pMX-Sox2 retrovirus. Percentages are SOX2+ cells from 100 nuclei (DAPI). Right: counts for N+ and N+E+ colonies following 7 days doxycycline treatment after pre-infection with pMX-Flag (Ctrl) or pMX-Sox2 (Sox2). Scale bar, 50 μm.