Cdx genes distinctly affect hematopoietic progenitor formation during EB differentiation. (A) Quantitative RT-PCR analysis of inducible Cdx1, Cdx2, and Cdx4 gene expression in differentiating day 4 EBs assessed 8 hours after exposure to 1 μg/mL doxycycline (dox). Expression is presented relative to expression level of each gene in noninduced day 4 EBs harvested at the same time point. Normalization to the housekeeping gene β-actin has been performed for each individual sample. Data are representative of multiple independent experiments.(B) Immunoblot for Cdx1, Cdx2, and Cdx4 protein levels in day 4 iCdx1, iCdx2, or iCdx4 EBs collected 8 hours after exposure to 1 μg/mL doxycycline (+). The multiple bands detected by anti-Cdx2 represent distinct phosphorylation forms.41 Control samples (−) represent EBs of the same ESC line, harvested at the same time point, that have not been exposed to doxycycline. Data are representative of multiple independent experiments. iCdx1, iCdx4, and iCdx2 EBs were exposed to doxycycline during the indicated windows of differentiation, dissociated at day 6 of differentiation, and then plated into secondary assays with or without continued exposure to doxycycline. The effect of Cdx gene induction on OP9 colony forming potential (C) and hematopoietic progenitor potential (D; CFU formation) was assessed. To pool and average data from multiple experiments (each data point denotes between 3 and 8 replicates), the data are presented as fold change in colony potential relative to noninduced control. P values calculated using nonpaired, 2-tailed Student t test are depicted over bars.
