Clonal phenotype of nucleus pulposus cell lines. (A) Representative phase-contrast images of cobblestone, wavelike and tiny cell morphology of immortalized donor 4 (D4) nucleus pulposus (NP) cells. (B) Representative phase-contrast images of cobblestone, wave-like and tiny cell morphology of immortalized D5 NP cells. Images were captured upon confluence in maintenance or differentiation medium. The percentage of the total amount of clones per phenotype is indicated below the images. Black bars in = 20 μm. (C) Scatterplot (top) of relative Brachyury T expression levels in immortalized NP clones. The horizontal bar represents the average T mRNA level of all six NP clones represented in the immunoblot images (bottom), which show T protein levels in NP clones. Each data point represents a triplicate measurement for an individual clone. The solid black bars represent the averages associated with specific clonal morphology. The dashed bar represents the average of six clones combined. The U-CH1 cell line was used as a positive reference for both mRNA and protein expression analyses.