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. Author manuscript; available in PMC: 2017 Dec 29.
Published in final edited form as: Cell Rep. 2017 Dec 26;21(13):3691–3699. doi: 10.1016/j.celrep.2017.12.004

Figure 2. Rlim-dependent and –independent XCI pathways exist in ESCs.

Figure 2

A) WT ESCs were split and in vitro differentiated in parallel either by EB or RA. Protein extracts were prepared at various time points during differentiation. The same Western blot was hybridized with antibodies against RLIM, OCT4 and β-actin. Note that Oct4 levels drop dramatically by 3d, while RLIM levels are not significantly downregulated by 6d. B) Comparison of Xist RNA levels in undifferentiated ESCs and after 6d RA-or EB-differentiation via ssRT-qPCR (control: E14 male ESCs; Xist levels of undifferentiated WTp cells are set to 1). Data represent three independent experiments. Note that Xist levels are < 3× higher in EB-differentiated WTp ESCs when compared to RA-differentiation, but > 50× for all RlimKOp ESCs. C) Formation of Xist clouds in (6d) EB-differentiated RlimKOp ESCs in RNA FISH using Xist as probe. Representative images are shown. D) Summary of three independent experiments as shown in (C). Xist clouds in ESCs of 10 EBs were evaluated with >100 cells counted per EB. Error bars indicate standard error of the mean (SEM).