QPCR analysis shows activation by Smad1 of hemangioblast-associated genes. The iSmad1 EBs were left untreated or treated with doxycycline from day 2 to 2.25. Age-matched untreated and treated samples were harvested at day 2.25, day 2.5, day 3, or day 4. The cDNA was generated from isolated RNA and QPCR was performed. The data were processed using the 2^-ΔΔCT method.26 The median of each sample was normalized to its respective age-matched untreated control and the median average (n = 3) was graphed as fold change in RNA expression. The QPCR analysis was performed for Smad1, Brachyury, Gata2, Scl, Runx1, Flk1, and Vegf, as indicated. Error bars indicate the SEM and the asterisks indicate a P < .01 compared to the no dox samples.