Fig 2: Functional expression of NMDAR in HEK293 cells using baculovirus. (A) NR1 and NR2A protein levels in HEK293 cells transduced with baculovirus. HEK293 cells were transduced in the presence of 1 mM Ketamine with different amounts of baculovirus encoding human NR1 and NR2A. Cells were harvested 16 h after transduction, and NR1 and NR2A protein levels were analyzed by immunoblotting. Representative images are shown. Molecular weight standards are indicated. NR2A shows an unspecific band at ~150 kDa. (B) Cell viability of HEK293 cells 16 h after transduction with different amounts of baculovirus encoding NR1 or NR2A. Cell viability was assessed using CellTiterGlo reagent. Data was normalized to non-transduced cells and represents the mean ± standard error of the mean (SEM) of three experiments. (C,D) Cell viability of HEK293 cells 16 h after transduction with baculovirus encoding NR1 or NR2A with concurrent treatment with MK801 (C) or Ketamine (D). Data was normalized to non-transduced cells in the presence of DMSO and represents the mean ± SEM of three experiments. (E) Model for lack of NMDAR activity in functional assay after protection with MK801 or Ketamine. NMDAR-ligands contained or secreted by the cells into the media activate NMDAR and lead to excitotoxicity and desensitization of the channel. Protection with MK801 or Ketamine prevent excitotoxicity, however, both compounds are difficult to wash out and do not prevent occupation of the ligand binding sites with endogenous ligand and prevent NMDAR activity in functional assays.

Fig 3: Downregulation of excitatory receptor and postsynaptic transmission after BPA exposure. A,B) Representative immunoblot and corresponding densitometric analysis showing the ratios of expression amounts of NMDA receptor subtypes (NR1, NR2A, and NR2B to GAPDH (#p > 0.05, *p < 0.05, and #p > 0.05, respectively) and AMPA receptor subtypes (GluR1 and GluR2) to GAPDH in control and juvenile BPA treated groups (n = 8 rats per group, *p < 0.05 and p > 0.05, respectively); C,D) Excitatory synaptic transmission in BPA-treated brain slices (10 × 10-6 m) was assessed by recording NMDA and AMPA receptor mediated current in the hippocampal CA1 areas, as shown by decreased EPSCNMDA, not EPSCAMPA, after acute BPA treatment (n = 8 rats per group, *p < 0.05 and p > 0.05, respectively).

Fig 4: Representative diagram showing the orientation of datumetine binding to NMDAR generated from the simulation software. Datumetine interacts with NMDAR at Tyr144.

Fig 5: 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