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. Author manuscript; available in PMC: 2014 Dec 5.
Published in final edited form as: Cell Stem Cell. 2013 Dec 5;13(6):676–690. doi: 10.1016/j.stem.2013.10.008

Figure 5. Thoc5 regulates the exit from the pluripotent state.

Figure 5

(A–B) Expression of Thoc5 and pluripotency genes during ESC differentiation induced by LIF withdrawal. Protein expression was determined by western blot (A). mRNA expression was determined by RT-qPCRs (B). Values were plotted as mean ± SEM from three independent experiments. (C) Thoc2 RIP after LIF withdrawal. RIP was carried out 24 hours after LIF withdrawal. Immunoprecipitated mRNAs were quantitated by RT-qPCRs and normalized to Input, and plotted as mean ± SEM from three independent experiments. (D) Effect of Thoc5 overexpression on Thoc2 interaction with the pluripotency gene mRNAs. RIP was carried out in the GFP (GFPOE) or Thoc5 overexpression (Thoc5OE) ESCs 24 hours after LIF withdrawal. Immunoprecipitated mRNAs were quantitated by RT-qPCRs and normalized to the input, and plotted as mean ± SEM from three independent experiments. *: p < 0.05. (E) Effect of Thoc5 overexpression on pluripotency gene expression. Expression of pluripotency gene proteins in GFP (Ind-GFP) or Thoc5 overexpression ESCs (two different lines Ind-Thoc5-1 and Ind-Thoc5-2) at the indicated time points during LIF withdrawal was determined by western blot. (F–H) Effect of Thoc5 overexpression on ESC self-renewal. (F) GFP (Ind-GFP) or Thoc5 (Ind-Thoc5-1, Ind-Thoc5-2) overexpression ESCs were cultured without LIF for the indicated amount of time, and the different types of colonies formed were counted and plotted as percentage of total. Values are plotted as mean from three independent experiments. (G) GFP (GFPOE) or Thoc5 overexpression (Thoc5OE) ESCs were cultured without LIF for the indicated amount of time. The expression of the differentiation markers was determined by RT-qPCRs and plotted as mean ± SEM from three independent experiments. (H) GFP (GFPOE) or Thoc5 (Thoc5OE) overexpression ESCs were cultured without LIF for 48 hours, and then re-plated at clonal density in ESC culture conditions. The number of ESC colonies formed 8 days later was counted and plotted as mean ± SEM from three independent experiments. Representative images of differentiated and undifferentiated colonies are shown at the top. See also Figure S5, S6.