Activated Notch1 Target Genes during Embryonic Cell Differentiation Depend on the Cellular Context and Include Lineage Determinants and Inhibitors (original) (raw)
Figure 10
Validating the Notch1 target genes Sox9, Pax6, Id4, Runx1 and Myf5.
Notch1 signaling was induced in ESC and differentiating embryoid bodies by addition of OHT (500 nM). (A) Immunoblot of EB5 NERT ESC (NERT ESCs) and EB5 NERT differentiated embryoid bodies (NERT Mes. 5d) with Sox9, Pax6 and Id4 antibodies. One representative example of three independent experiments is shown. (B) Mesodermal differentiation of Runx1-Venus-NERT ESC or Runx1-Venus control ESC and analysis of the percentage of venus+ and Flk1+ cells by FACS. Error bars indicate standard deviation for three independent experiments. (C) Mesodermal differentiation of Myf5-lacZ-NERT or Myf5-lacZ control ESC and detection of LacZ expression. One representative example of three independent experiments on day 5 is shown. Virtually identical results were obtained when the cells were analyzed 4 or 7 days, respectively, after induction of differentiation. Addition of OHT at day 3 of embryoid body culture similarly yielded in an increased number of lacZ positive cells at days 4, 5 and 7 in the presence of OHT after induction of differentiation.