Fig. 3

Triple activation of GR, ERα, and RARα enhanced neuronal differentiation in BE(2) cells. A Immunofluorescence staining after ligand treatment. Green = βIII-tubulin; red = SCG2; blue = DAPI. Scale bars represent 20 μm. B Western blot analysis of neural differentiation markers in BE(2)-GR + EV and BE(2)-GR + ERα cells following incubation with ligands. β-actin was used as loading control. Molecular weight markers shown to the left. C RT-qPCR of NEFL, TRKA, DLG2, NES, SOX2, and MYCN in BE(2)-GR+EV and BE(2)-GR-ERα cells following ligand treatment. B2M was used as housekeeping gene. Statistical analysis: t-test with *, **, ***, and **** indicating p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. The brackets in the DLG2 graph represent significance of all the experimental conditions versus their controls. Data of the mRNA expression fold is presented as mean ± SD of a minimum of three independent replicates. In all experiments, 10 nM E2, 100 nM DEX, or 0.5 µM ATRA were used with ethanol as control for E2, DEX, and E2 + DEX, DMSO for ATRA, and ethanol + DMSO for the triple combination. All experiments were carried out during seven days and performed at least three times