Figure 2.

Ligand-induced Notch signaling significantly induces Hey1 and HeyL expression, but only constitutively expressed Hey1 blocks myogenesis. A) 6-well plates were coated with 3 ml of ligand-containing supernatant per well. C2C12 cells were plated on Fc-Dll4 or Fc-control ligand and propagated for 48 hours in growth medium (GM). Hey1, Hey2 and HeyL RNA levels were determined by quantitative RT-PCR using 18S as a loading control. Expression levels (x) for individual genes were computed from ΔCt values (relative to 18S) according to the formula (x) * 2^ΔCt = (c), where c is an arbitrary constant, and plotted as the average +/− standard deviation of three replicate samples. B) C2C12 cells were stably transduced with parental retrovirus (pBABE-puro) or retroviruses expressing FLAG-tagged Hey1 or HeyL and analyzed for expression of the indicated cDNAs by RT-PCR. RT, reverse transcriptase. C) Transduced cells were propagated in GM, shifted to DM and analyzed for expression of Myogenin, MHC or FLAG-tagged proteins after 1, 2, or 4 days by Western immunoblotting using β-tubulin as a loading control. D) Human myoblasts were grown on Fc-Dll4-coated or Fc-control-coated plates and switched from GM to DM for 24 hours as indicated. Myogenin protein was assessed by Western immunoblotting using β-tubulin as a loading control, and Hey1 RNA was determined by RT-PCR using GAPDH as a loading control.