FIG. 4.
γ-Secretase inhibition of embryoid bodies (EBs) differentiated using the 2 induction strategies. (A) Frequency of multipotent hematopoietic colonies (CFU-Mix, CFU-GM, Ery/Mac) per 1 × 105 input embryonic stem (ES)-derived cells differentiated to 6 days on gelatin, AM14.1C4 or OP9 in the presence (+) or absence (−) of γ-secretase inhibitor (GSI), added between Days 4 and 6. One representative experiment of 3 is shown. (B) Quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed a significant reduction in Hey1 gene transcript in EBs in the presence of γ-secretase inhibitor (GSI). (C) γ-Secretase inhibition of HOXB4-ERT2 EBs differentiated on gelatin. Cells were either noninduced or induced with tamoxifen in the presence (+) or absence (−) of GSI, added between Days 4 and 6. The fold induction in CFU-Mix, CFU-GM, and Ery/Mac colony numbers over noninduced control HOXB4-ERT2 EBs is shown (±SD between experiments). (D) Quantitative RT-PCR showing an increase in Hey1 gene transcript after HOXB4 activation which was inhibited in the presence of the γ-secretase inhibitor (GSI). (E) Quantitative RT-PCR showing the expression pattern of Jagged1 in control CGR8 EBs or HOXB4-ERT2 EBs differentiated in the presence (+) or absence (−) of tamoxifen, added from Days 2 to 4.