Fig. 5

Effects of pharmacological modulation of NMDAR function on chondrogenesis. a. Metachromatic cartilage areas in 6-day-old high density colonies were visualised with DMMB dissolved in 3% acetic acid (pH 1.8). Metachromatic (purple) structures represent cartilaginous nodules formed by multiple cells and an ECM rich in polyanionic GAGs (n = 3 for each experimental condition). Original magnification was 2×. Scale bar, 1 mm. Optical density (OD₆₂₅) was determined in supernatants of 6-day-old cultures (n = 3 for each experimental condition) containing toluidine blue extracted with 8% HCl dissolved in absolute ethanol. Representative data out of 3 independent experiments. Statistically significant (*P < 0.05) differences in extinction (OD₆₂₅) of samples for TB relative to the respective vehicle control are marked. b. Protein expression and phosphorylation status of the master chondrogenic transcription factor Sox9 in 3-day-old cultures (n = 3 for each experimental condition). Optical densities of bands from 3 experiments were pooled and presented as bar graphs ± SEM, along with representative membrane images from a single experiment. The representative membrane images are out of 3 independent experiments, each showing a similar expression profile. Data normalization was performed by normalizing each data to the optical density values of the loading control (GAPDH) for the same experimental condition, and then to the control cultures (dark grey columns). Significant (*P < 0.05) alterations in protein levels of the mean signal densities from 3 experiments relative to the respective controls (mean) are marked