Fig. 4.

Arf and p53–ER promote expression of ExEn marker proteins. (A) Lysates of WT MEFs (left lane) or from cells transduced with a vector encoding Arf exon-1β sequences (center lane) or with a naked control vector (right lane) were immunoblotted with antibodies directed to p19^Arf (Upper) or to the ER cassette (Lower). The endogenous p19^Arf protein was detected in all three samples, whereas the 50-kDa minigene-coded fusion protein was detected only in the center lane. The asterisk indicates a staining artifact. (B) Arf-null iPS cells transduced with the vector encoding the Arf–exon-1β minigene were subjected to qPCR analysis. Primers that amplified the ER moiety confirmed generally equivalent levels of vector-coded RNA expression in cells treated with different concentrations of tamoxifen (TAM; see legends). Dab2 and Gata4 mRNAs in transduced Arf-null iPS cells were induced, and Nanog expression was decreased in response to minigene expression and tamoxifen treatment. Error bars, mean ± SEM (n = 3 experiments). (C) Phase contrast micrographs reveal that EBs derived from p53-null iPS cells (Right) fail to express a halo of ExEn cells visualized in WT EBs (Left). (D) Like day-4 EBs derived from Arf-null iPS cells, p53-null iPS cells exhibited reduced Dab2 protein levels compared with WT iPS controls. (E) Although p19^Arf protein expression was detected at the periphery of day-4 EBs derived from p53-null iPS cells, neither Gata4 nor Dab2 were detected. Instead, Oct4-positive cells were observed at the periphery. (Scale bars, 200 µm.) (F) Arf-null iPS cells were transduced with a retroviral vector expressing the HRas–G12V/T35 mutant. Cells engineered to coexpress p53–ER^TAM (Center and Right) were treated with tamoxifen (Right) or not (Center), and Dab2 expression was quantified by flow cytometric analysis. The percentages of cells that exhibited increased Dab2 expression are noted.