Fig. 3.

Ras/Erk signaling promotes Arf-dependent ExEn differentiation. (A) WT and Arf-null iPS cells (indicated at left) were transduced with vectors expressing the indicated Ras mutants or with a control (Ctl) vector, selected for puromycin resistance in medium containing LIF, and induced to differentiate for 4 d in the absence of LIF. A constitutively active Ras effector mutant (G12V/T35S) that signals to Erk promoted ExEn cell formation from WT, but not Arf-null, iPS progenitors, an effect blocked by a dominant-negative mutant (HRas–S17N). Phase contrast images are shown. (Scale bars, 200 µM.) (B and C) Quantitative PCR (qPCR) analysis (B) and immunoblotting (C) confirmed Dab2 mRNA and protein up-regulation in WT cells infected with the G12V/T35S Ras mutant (abbreviated as T35S). (D) Activated phospho-Erk was detected at the periphery of both Arf-null and WT day-4 EBs generated from the different iPS progenitors. (Scale bars, 200 µm.)