Fig. 2

Characterization of the BZ cell line. BZ cells are cultured in DMEM supplemented with 1% FBS unless stated otherwise. a, BZ cells present with neuronal features. BZ cells display an elongated morphology harboring multiple short cellular processes, brightfield microscopy (magnification x20, upper left panel). Immunolabeling images (x40) with negative control secondary antibody and DAPI counterstaining (upper right panel), β-tubulin III (lower left panel) and merged DAPI and β-tubulin III (lower right panel). b, GR is expressed in BZ cells. Immunocytochemistry of GR staining on BZ cells grown in 10% FBS, showing a homogenous expression of GR in this cell line. GR, upper panel, DAPI, middle panel, Merged, lower panel (c), Analytical RT-PCR of GR, BDNF, NMDA receptor ζ (NMDAR) and MAP2 mRNA expression (30 cycles of amplification with DreamTaq (Thermo Scientific)) on BZ cells compared with neurons derived from mouse embryonic stem cells (ES) and mouse brain. d, Western Blot on BDNF protein in BZ cells, primary cultures of mouse hippocampal neurons (PCN), and mouse brain, showing that BDNF protein was clearly detected in all of the 3 samples. e, Immunoprecipitation in BZ cell lysates with anti-GR antibody, further demonstrating GR expression, MW: molecular weight markers in kDa, IgG: rabbit immunoglobulins, GR band is around 105 kDa. f, DEX was able to transactivate GRE2.Luc, indicating the presence of a functional GR in BZ cells. Transfection of BZ with a PGL3-Gre2.Luc plasmid under DEX dose responses (10^-9 to 10^-6 M, n = 8, Mean ± SEM); Vect, GRE2.Luc; Veh set at 1; * P < 0.05, ** P < 0.01, Mann Whitney U-tests. g, Increased expression of neuronal marker MAP2 transcripts at low FBS (1%) concentration. MAP2 (higher panel), GR (middle panel) and BDNF (lower panel) mRNA expression in BZ cells under various FBS concentrations from 1 to 10%; n = 6, Mean ± SEM; ** P < 0.01 vs 1% condition, Mann Whitney U- tests