Fig. 7

Effects of transient gene silencing using of the GRIN1 siRNA on chondrogenesis. a. Metachromatic cartilage areas in 6 or 10-day-old high density colonies (n = 3 for each experimental condition on each day) were visualised with DMMB. Original magnification was 2×. Scale bar, 1 mm. Optical density (OD₆₂₅) was determined in supernatants of 6 or 10-day-old cultures (n = 3 for each experimental condition on each day) containing extracted toluidine blue. Statistically significant (*P < 0.05) differences in extinction (OD₆₂₅) of samples for TB are marked. b. mRNA expression of the GRIN1 gene following gene silencing with siRNA on culturing days 3 and 10 (n = 3 for each experimental condition on each day). Non-targeting (NT) siRNA was used as a control, and GAPDH was used as a reference gene. c. Proliferation rate on days 3 and 10 in micromass cultures (n = 3 for each experimental condition on each day; average ± SEM). Significant (*P < 0.05; ***P < 0.0001) changes of proliferation rate relative to the non-targeting (NT) control (on day 3) are marked. d. Protein expression of the GluN1 subunit, as well as expression and phosphorylation status of Sox9 in 3- and 10-day-old cultures (n = 3 for each experimental condition on each day). GAPDH was used as an internal control. 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 the density date of the gene silenced cultures to the optical density values of the loading control (GAPDH) for the same experimental condition, and then to the NT control cultures (dark grey columns). Significant (*P < 0.05) alterations in protein levels of the mean signal densities from 3 experiments relative to the NT control (mean) on the same culturing day are marked. All panels show representative data out of 3 independent experiments showing the same tendency of changes