Figure 2.

RAS Repression by LIF Prevents Early Differentiation

(A–C) mESCs were differentiated in the absence of LIF for 72 hr and in the presence of the RAS inhibitor farnesylthiosalicyclic acid (RASi, at the indicated concentrations or 75 μM if not mentioned) or vehicle (Veh) as control. (A) The relative RAS-GTP or total RAS protein levels were analyzed in cells treated with the indicated concentration of RASi. (B) Western blots of OCT4 and AKT (loading control). (C) mESCs or Rex1-YFP-expressing mESCs were differentiated in the absence of LIF and in the presence of RASi (75 μM) or Veh for 72 hr. Cells were stained for SSEA1 and OCT4, and YFP fluorescence was measured by flow cytometry. The table shows mean fluorescence of each staining or YFP.

(D–F) mESCs were infected with shRNAs against the indicated RAS isoform or control shRNA and the levels of the relevant RAS isoforms were examined by real-time PCR analysis (D) and by western blot (E). Western blot of OCT4 and AKT of infected mESCs differentiated in the absence of LIF for 72 hr (F).

(G and H) mESCs were transfected with a plasmid encoding for GFP fused to H-RAS (GFP-H-RAS) or GFP as control. GFP-positive cells were sorted by flow cytometry, lysed, and immunoblotted using anti-GFP antibody (G) and pluripotency markers (H). AKT or ERK were used as a loading control.

(A, B, and E–H) Values represent densitometry analysis of at least three independent experiments. Data shown are mean ± SD from three independent experiments. ^∗p < 0.05 statistically significant by Student's t test.