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Fig. 3
D3 Antagonizes Hh Signaling via Interaction with the Extracellular Domain of Smoothened
(A) Diagram depicts the sites of action of Hh pathway modifiers cyclopamine, SAG, and 20-OHC on the Smoothened receptor. The presumptive site of D3 action (red) is shown.
(B) FACS of Gli-reporter embryos showed D3 decreased Hh activity (∗p = 0.002). SAG co-treatment (5 μM) did not alter the effect of D3, while 20(S)-OHC (10 μM) partially blocked Hh inhibition (∗∗p = 0.040) (5 embryos/sample × 4 replicates/condition). Error bars, mean ± SD.
(C) Co-treatment with 20(S)-OHC corrected D3-mediated reductions in ptc2 (∗p = 0.019) and runx1 (∗∗p = 0.003) by qPCR (40 pooled embryos/condition × 3 replicates). Error bars, mean ± SD.
(D) 20(S)-OHC restored runx1 expression in D3-treated embryos. Scale bar, 100 μm.
(E) Qualitative phenotypic distribution of embryos from (D) scored for runx1 (n > 20 embryos/condition).