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).