Figure 4From: Novel immortal human cell lines reveal subpopulations in the nucleus pulposus 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.Back to article page