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Supplementary Material

JCS084699 Supplementary Material

Files in this Data Supplement:

  • Supplemental Figure S1 -

    Fig. S1. Loss of Cyp26b1 in the limb leads to ectopic activation of the retinoid signalling pathway. (A) Alcian Blue and Alizarin Red skeletal staining of E18.5 Cyp26b1+/+ and Prrx1Cre-/Cyp26b1fl/fl embryos demonstrates that there is no difference between wild-type controls in the two transgenic mouse lines. Scale bar: 2.5 mm. (B) X-gal staining of E11.5 and E13.5 limbs from wild-type and Cyp26b1−/− mice expressing the RARE-lacZ transgene. Scale bar: 2 mm. (C) qPCR analysis of Cyp26b1 and Rarb expression in forelimb proximal (FP) and distal (FD) and hindlimb proximal (HP) and distal (HD) regions from E11.5 limbs. Single embryos were analyzed and repeated with similar results. (D) qPCR analysis of Cyp26b1, Crabp2, Fgf18, Twist1 and Vegfa expression in PLM cultures after 1 or 3 days of culture. Error bars represent 1 s.d. Significance was evaluated in comparison to wild-type controls on the same day and is represented as follows: *P<0.05; **P<0.01; #P<0.001. nd, 40 cycles of qPCR and transcript not detected.

  • Supplemental Figure S2 -

    Fig. S2. Co-expression of Rara or treatment of ketoconazole does not directly affect SOX9 activity. Wild-type cultures were co-transfected with Col2-LUC with and without expression plasmids for Sox9 and/or Rara as indicated. After 24 hours, cultures were treated with keto (1 µM) and luciferase activity was measured 24 hours later. Significance was evaluated in comparison to the ketoconazole untreated control and is represented as follows: **P<0.01.

  • Supplemental Figure S3 -

    Fig. S3. Modulation of RA signalling preferentially impacts the early stages of chondrogenesis and establishment of a chondrogenic template. PLM cultures from wild-type embryos were treated with various concentrations of ketoconazole at the indicated time points (D1, D3 or D5) and stained with Alcian Blue (left column) or for alkaline phosphatase (right column) at day 8. Scale bar: 1 mm.

  • Supplemental Table S1 -

    Table S1. Primer and probe sequences generated for qPCR