Extracellular matrix molecules in nucleus pulposus differentiation. (A) Extracellular matrix (ECM) properties in responsive nucleus pulposus (NP-R) clones (left column; R) and nonresponsive nucleus pulposus (NP-nR) clones (right columns; nR). Sulphated glucosaminoglycan (GAG; left graph) and hydroxyproline (right graph; OH-pro) quantities in four representative NP-R clones (108, 114, 115 and 124; grey circles) and four NP-nR clones (102, 105, 113 and 119; black circles) at baseline (t = 0), at 7 days in differentiation medium (Dmed) and at 7 days in maintenance medium (Mmed). Statistical significance was assessed by Student’s t-test. *P < 0.05; **P < 0.01; n.dct., Not detectable. Full list of P-values is provided in Additional file 1: Table S3. (B) Comparison of marker expression on monolayer (mono; uncoated polystyrene), Matrigel-coated (M.gel) and aggrecan- coated (ACAN) cultured NP cell clones. Gene expression levels of SRY-box 9 (SOX9), COL2A1, ACAN and cartilage oligomeric matrix protein (COMP) are depicted. Grey bars correspond to NP-R115, and black bars to clone NP-nR105. Gene expression was normalized to β-ACTIN mRNA levels. Data are expressed as fold changes relative to expression levels at baseline (t = 0) (Mmed). Statistical significance was assessed by Student’s t-test. Full list of P-values is provided in Additional file 1: Table S4. *P < 0.05; **P < 0.01; n.dct. = not detectable. (C) Phase-contrast images of representative NP-R clones and NP-nR clones on Matrigel-coated plates at 36 hours postplating. (D) Phase-contrast images of representative NP-R clones and NP-nR clones on monolayer plastic or ACAN coating at 72 hours postplating. Black bars = 20 μm.