concentrations of cyclopamine (B-E). At the end of the culture cyrosections were collected at 6 mm thickness and stained using a specific antibody for Gli1 (red). (A) shows Gli1 expression in all the regions of the vehicle treated control IVDs. (B) to (E) show progressive reduction of Gli1 expression with increasing doses of cyclopamine (50–250 mM respectively). Scale bars indicate magnifications used. Red = expression of Gli1. NP = nucleus pulposus, AF = annulus fibrosus, EP = end plate. (TIF) Figure S2 Doxycycline treatment of tetO-cre transgenic mice shows no change in the phenotype of the NP (A), AF (B), and EP (C) cells. Immunostaining for differentiation markers used for comparison are as shown in Figures 3, 4 and 5 in the manuscript. (A) Shows that the expression of Gli1, ptch1, Shh, Bra, Sox9, chondroitin sulfate, and collagen 1 continues to be expressed in the NP cells. (B) Shows the expression of Gli1, ptch1, Sox9, collagen 1, collagen 2, chondroitin sulfate and keratin sulfate are present in the AF cells. (C) Shows histology and immunostain- ing for Gli1, Sox9, collagen 2 and Hh in the EP cells. Scale bars indicate magnifications used. IAF = inner annulus fibrosus, OAF = outer annulus fibrosus. Red = expression of specific protein, Blue = cell nuclei stained with POPO-3, green = general counterstain with wheat germ agglutinin. EP = end plate, GP = growth plate. (TIF) Figure S3 Shows that P4 IVDs cultured for five days (P4 t5) after removal of the hypertrophic zone of the vertebral growth plates (- HZGP) continue to have normal histology (A) and expression of the differentiation markers (B–G) shown in Figure 6 in the manuscript. (A) Shows the reticular network of NP cells and the References 1. Deyo RA, Tsui-Wu YJ (1987) Descriptive epidemiology of low-back pain and its related medical care in the United States. Spine (Phila Pa 1976) 12: 264–268. 2. 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