The mesodermal developmental program is accelerated in response to mMix. (A) General experimental protocol for induction and analysis of i-Mix EBs cultured with or without DOX. DOX (0.1 μg/mL) was added on day 1 after plating under differentiation conditions. (B) Semiquantitative RT-PCR analysis of gene expression in i-Mix EBs cultured with or without DOX. EBs were harvested on days 2.0 (lanes 2, 12), 2.3 (lanes 3, 13), 2.6 (lanes 4, 14), 3.0 (lanes 5, 15), 3.3 (lanes 6, 16), 3.6 (lanes 7, 17), 4.0 (lanes 8, 18), 4.3 (lanes 9, 19), 4.6 (lanes 10, 20), and 5.0 (lanes 11, 21). NT (lane 22) indicates minus template control. Expression of T was transient, preceding activation of Flk1 and c-kit. The differentiation program in DOX-induced i-Mix cells was accelerated (see text). (C) Quantitative real-time RT-PCR analysis of primitive streak and early mesodermal gene expression in i-Mix EBs cultured with or without DOX. Expression was normalized to that of a housekeeping gene (for this figure, Gapdh) and expressed as a ratio (see “Materials and methods”).