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Fig. 2. (Top) Analysis of differentiation of C2C12 and satellite cells. C2C12 and
satellite cells were differentiated in the presence and absence of BMP4 and/or
L-685,458. The number of MHC-positive cells and total number of DAPI-positive
cell nuclei were counted in randomly selected microscope fields after
immunostaining. The percentage of MHC-positive cells compared with the number
of DAPI-stained nuclei was calculated. To the right are examples of
multinucleated C2C12 and satellite cells stained for MHC. (A-H) Analysis of
Hes1 and Hey1 mRNA expression in response to BMP4 and Notch
signaling. The amount of Hes1 (A) and Hey1 (B) mRNA, as
measured by quantitative PCR, in C2C12 cells after addition of L-685,458
and/or BMP4. (C) An experiment similar to that in A and B, but measured at
both 0 and 5 days after induction of differentiation. (D) Comparison of
changes in expression of Hey1 and Runx2 in response to BMP4
and/or L-685,458. (E) Western blot analysis of MHC and MyoD protein expression
at various time points after induction of C2C12 differentiation. Below is a
quantitative PCR experiment demonstrating changes in MyoD expression
in response to BMP4 and/or L-685,458. (F,G) The amount of Hey1 (F)
and Hes1 (G) mRNA, as measured by quantitative PCR, in satellite
cells after addition of L-685,458 and/or BMP4. (H) The amount of Hey1
mRNA, as measured by quantitative PCR, in neural stem cells cultured as
neurospheres under normal conditions or after addition of BMP4.
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