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. Author manuscript; available in PMC: 2007 Jan 3.
Published in final edited form as: Nat Cell Biol. 2005 Jun 19;7(7):698–705. doi: 10.1038/ncb1272

Figure 5.

Figure 5

Hedgehog signaling decreases D2 by inducing WSB-1. (A) D2 activity was measured in HEK-293 cells transiently expressing D2 co-cultured for 24 h with an equivalent number of HEK-293 cells transiently expressing Shh or D10 vector. Vehicle or 5μM cyclopamine were used as indicated. iWSB-1 was employed as indicated. D2 was quantified by western blotting and numbers match gel lanes to bars. *p<0.05 vs. vehicle-treated cells (ANOVA). (B) Time-course of D2 activity and WSB-1 mRNA levels by real-timer PCR in cells prepared as in (A). Results are expressed relative to time zero and the loss of D2 activity negatively correlates with time (p<0.0001). In A-B, results are the mean ± SD of 4-6 samples. (C) WSB-1 in situ hybridization of chicken tibiotarsi from day 12 chicken embryos cultured in serum-free medium containing 740 pM T4 and treated for 24 h with vehicle, Shh (4 μg/ml) or cyclopamine (5μM). The bone area shown analyzed is labeled as PC/PO in the schematic of the growth plate. At least 4 tibias were examined for each experimental condition with similar results. Hybridization with the sense probe was negative (not shown). (D) D2 activity and different mRNA levels in tissues of tibiotarsi isolated and processed as in (C): perichondrium/periosteum (PC/PO), hypertrophic chondrocytes (HC), and bone epiphysis (EP). Results are the mean ± SD of 4 embryos. *p<0.05 vs. untreated tibiotarsi by Student's t-test. (E) D2 activity and mRNA levels in day 8 chicken embryo hind limb misexpressing Ihh. The uninfected tibia was used as control. Results are the mean ± SD of 3 embryos. *p<0.05 vs. uninfected tibial tissue by Student's t-test. (F) D2 activity and different mRNA levels in tissues of tibiotarsi isolated and cultured as in (C), and treated with 20 nM rT3 for the indicated times. Results are the mean ± SD of 4 embryos. *p<0.05 by Student's t-test vs. untreated tibiotarsi. (G) PTHrP mRNA levels in tibiotarsi isolated and cultured as in (C); treatments were 20 nM rT3 and/or 5μM Cyclopamine. Results are the mean ± SD of 2 embryos. *p<0.05 vs. untreated tibiotarsi by one way ANOVA.