Fig. 6.

HS antagonism modulates chondrogenic response to BMP2. (A–H) Representative mouse limb bud cell micromass cultures stained with Alcian blue on day 6 showing that cartilage nodule formation is stimulated by Surfen (C) or rhBMP2 (F) over respective controls (A and E). The pro-chondrogenic effects of Surfen are prevented by Noggin co-treatment (D), but are further enhanced by rhBMP2 co-treatment (H). (I) Id1-luc reporter assays indicating that treatment with either rhBMP2 or Surfen increases BMP signaling while rhBMP2/Surfen co-treatment stimulates it over 10-fold; Noggin treatment reduces it. Values are means+S.E. of three experiments. (J) Immunoblots showing that the levels of phosphorylated Smad1/5/8 are increased by treatment with rhBMP2 or Surfen by 4 h and remain high, while total Smad1 levels remain steady. (K–M) RT-PCR analysis showing that BMP2, BMPRI and BMPRII expression increases over time after initiating rhBMP2 or Surfen treatment. Values are means±S.E. of three experiments. Scale bar, 3 mm in A.