Fig. 3. Maintenance of stable Myc at physiological levels sustains self-renewal in the absence of LIF/Wnt3a CM. Myc expression was driven by the CAG promoter as a fusion with the steroid binding domain of the estrogen receptor (ER) for inducible activity and coupled to a puromycin resistance gene (puro^R) by an internal ribosome entry site (IRES). (A) Flow cytometry profiles of SSEA1 reactivity on the surface of mycER or myc^T58AER cells grown up to 12 days in the presence of LIF (+LIF) without 4OHT (-4OHT), or without LIF (-LIF) in the presence or absence of 4OHT. (B) Colony morphology of myc^T58AER transfected ES cell colonies grown in the presence or absence of 4OHT 6 days after LIF withdrawal. (C) MycER (circles, squares) and myc^T58AER ES cells (triangles) were grown in varying concentrations of LIF (0-1x10³ units/ml), in the presence (black symbols) or absence (white symbols) of 4OHT. Colonies were assayed for AP activity every 3 days after LIF withdrawal in duplicate. MycER cells were also grown in the presence of 7.5 units/ml LIF with (closed square) or without (open square) 4OHT. (D) Immunoblot of cell lysate from the myc^T58AER transfected D3 ES cell line showing levels of endogenous Myc, myc^T58AER and Cdk2 after being probed simultaneously with anti-Myc and Cdk2 antibody, and then exposed to film for the same length of time. (E) RT-PCR analysis of Oct4, Rex1 and ß-actin transcripts in vector alone or myc^T58AER cells grown for 14 days in the presence or absence of LIF and 4OHT.