Fig 1: (a) Variation in growth differentiation factor-6 (GDF-6) levels with aging and intervertebral disc (IVD) degeneration. Levels of GDF-6, bone morphogenetic protein receptor type II (BMPRII), IVD nucleus pulposus (NP)-associated notochordal brachyury, CD24, and actin (used as a loading control) were examined by Western blotting in protein extracts from surgically obtained human IVD NP tissues. Samples were randomly selected (n = 12). (b) Validation of the IVD NP phenotype in cell clusters. Levels of IVD NP-associated notochordal brachyury and CD24 were evaluated by Western blotting in protein extracts from human IVD NP cells. Immunoblots shown are representative of in vitro human IVD experiments, as described below, with similar results. (c) Effects of growth differentiation factor-6 (GDF-6) treatment on CD24, aggrecan, and type II collagen expression in human IVD NP cells (Pfirrmann degeneration grades III and IV, n = 4). Cells were cultured in a monolayer medium, followed by seeding in a three-dimensional culturing system (Tapered Stencil for Cluster Culture [TASCL]) at 1.0 × 106 cells/TASCL to form cell clusters. After 48-h culture, cell clusters were treated with solvent as a control (group C, set as 1.0), GDF-6 treatment (100 ng/mL, group G), pro-inflammatory interleukin (IL)-1β (10 ng/mL, group I), or both GDF-6 and IL-1β (group G + I). Then, CD24 (purple), aggrecan (green), type II collagen (red), nuclear 4′,6-diamidino-2-phenylindole (DAPI) (blue), and merged signals of cell clusters on TASCL were obtained using a fluorescence microscope. Relative fluorescence intensity of 10 randomly selected cell clusters per image was measured. Data are the mean ± standard deviation. (n = 4). One-way analysis of variance with the Tukey–Kramer post-hoc test was used. Significant differences were set as * p < 0.050 and ** p < 0.010.