Marker expression in clonal subtypes. (A) Immunoblot analyses of collagen type II, α1 (COL2A1), SOX9 and collagen type II, α1 (COL1A1), in responder nucleus pulposus (NP-R) clones (left panels) and nonresponder nucleus pulposus (NP-nR) clones (right panels). Cell clones were incubated for 7 days with differentiation medium (Dmed; ‘D’ in figure) or maintenance medium (Mmed; ‘M’ in figure). The different subtype samples were run on the same gels to enable direct quantitative comparison. Three representative clones per NP subtype are shown. β-actin (bACT) was used as a loading control. Representative phase-contrast images of NP-R clones 108, 114 and 115 and of NP-nR clones 105, 113 and 119 are shown in the bottom row. Black bars = 20 μm. (B) (top right panel; continued in lower panels) Scatterplots of relative gene expression analysis of keratin 19 KRT19, Carbonic anhydrase XII (CA12), cluster of differentiation 24 (CD24), Forkhead box F1 (FOXF1), paired box 1 (PAX1), pleiotrophin (PTN) and cartilage oligomeric matrix protein (COMP) in six representative NP-R clones (104, 108, 114, 115, 121 and 124; grey symbols) and six representative NP-nR clones (102, 105, 110, 113, 116 and 119; black symbols) at baseline (t 0) (Mmed) or at 7 days of culture in Dmed. Gene expression was normalized to bACT mRNA levels. Log2-scaled expression data are presented relative to the average expression level of NP-nR at t 0. Each data point represents a triplicate measurement for an individual clone. The P-values were obtained by comparing the indicated experimental groups. Statistical significance was assessed by Student’s t-test. *P < 0.05; **P < 0.01. Full list of P-values is available in Additional file 1: Table S2.