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Fig. 6. Hh signaling induces osteogenesis in C3H10T1/2 cells. (A) Induction of AP
by N-Shh. Results are shown for triplicate samples from a representative
experiment. This applies to all AP assays that follow. (B,B')
Cell-autonomous induction of AP by the constitutively active Smo*.
(B) A fluorescence picture showing cells expressing GFP in the nucleus.
(B') A bright-field picture of the same field showing cells stained for
AP activity. Numbers indicate cells expressing both GFP and AP. Asterisk
indicates a cell expressing GFP but not AP. (C,C') Bone nodule formation
induced by the constitutively active Smo*. Retroviruses encoding
Smo* induced bone nodules (arrows) stained positive by Alizarin Red
(C'), whereas a control virus did not (C). (D-G) Expression of
Col1a1 (D), Bsp (E), Runx2 (F) or Osx (G)
in Hh-treated (red bars) versus control cells (grey bars) by real-time PCR.
The mRNA levels were normalized to the level of glyceraldehyde-3-phosphate
dehydrogenase (GAPD) mRNA. Results are shown for triplicate samples from a
representative experiment. This also applies to other real-time PCR
experiments that follow. (H,I) Effects of cycloheximide on Hh induction of
Runx2 (H) and Osx (I) expression. Runx2 and
Osx mRNA levels were determined by real-time PCR and normalized to
Gapd for cells stimulated for 24 hours with N-Shh in the presence of
either DMSO (C) or 10 µg/ml cyclohexamine (CHX).
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