Journal: bioRxiv

Article Title: Antidepressants interact with sex steroid receptors and their intracellular signaling components

doi: 10.64898/2026.03.17.712321

Figure Lengend Snippet: Antidepressants rapidly activate ERα. (A) The relative change in ERα phosphorylation at Ser167 in MCF-7 cells compared with baseline control following treatment with S-ketamine (10 µM) and imipramine (10 µM) for 10 minutes. Phosphorylation levels were measured using a sandwich ELISA with luminescence readout. Estradiol (100 nM) served as the positive control for ERα activation. ( B) Inhibition of ERα by fulvestrant pretreatment eliminates antidepressant activation of ERα as measured by Ser167 phosphorylation. A 24-hour pretreatment of ERα with fulvestrant at 200 nM was followed by a 10-minute treatment while maintaining the concentration of ICI at 100 nM. Western blotting confirmed a lower amount of ERα protein after ICI pretreatment. ( C) MCF-7 cells were treated with S-ketamine (10 µM), imipramine (10 µM), or estradiol (100 nM) for 10 minutes. All treatments gave rise to an increase in the level of phosphorylated Ser167 compared to the baseline. Inhibition of membrane ERα by tunicamycin (15 µM for 30 min) prior to treatment with antidepressants and estradiol reduces the effect of antidepressants and estradiol. Western blotting exhibited no change in the amount of total ERα protein after pretreatment with tunicamycin. ( D) HSP27 in complex with ERα after pretreatment with tunicamycin (15 µM for 30 min) in various 10-minute treatment conditions. The levels of HSP27- ERα complex were measured using a sandwich ELISA with luminescence readout. Tunicamycin reduces the percentage of HSP27-ERα complex regardless of treatment conditions. The statistical significance is denoted as (ns: non-significant, *p < 0.05, **p < 0.01, *** p< 0.001, ****p < 0.0001).

Article Snippet: Imipramine hydrochloride (Sigma-Aldrich, I7379), fluoxetine hydrochloride (LKT Labs, F4780), and S-ketamine (Pfizer, 2750 Ballerup, Denmark) were used as antidepressants and 17-β estradiol (Sigma-Aldrich, E2758) served as the positive control in the study.

Techniques: Phospho-proteomics, Control, Sandwich ELISA, Positive Control, Activation Assay, Inhibition, Concentration Assay, Western Blot, Membrane

Journal: bioRxiv

Article Title: Antidepressants interact with sex steroid receptors and their intracellular signaling components

doi: 10.64898/2026.03.17.712321

Figure Lengend Snippet: Antidepressants selectively activate MAPK signaling and induce ERα-dependent gene expression in MCF-7 cells. (A) Quantification of MAPK phosphorylation (pMAPK/MAPK) by Western blot densitometry following treatment with vehicle, imipramine, S-ketamine, or estradiol (E2), in the presence or absence of the ERα degrader/antagonist fulvestrant and/or tunicamycin. Antidepressant treatment significantly increased MAPK phosphorylation, indicating enhanced MAPK pathway activation. (B) Total MAPK levels normalized to β-actin were unchanged across treatment conditions. Representative immunoblots for MAPK and pMAPK are shown above. (C) Quantification of Akt phosphorylation (pAkt/Akt) revealed no significant changes following antidepressant or E2 treatment, but there was a trend for a reduction by imipramine. (D) Total Akt levels normalized to β-actin were unaltered. Representative immunoblots for Akt and pAkt are shown above. One imipramine-treated sample was excluded from analysis due to an outlier pMAPK/MAPK ratio exceeding 1. Data from technical and biological replicates were pooled. (E–F) mRNA expression of the extranuclear-initiated ERα target gene LRRC54 (E) and the classical nuclear ERα target gene PgR (F) , quantified by qPCR following treatment with E2, imipramine, or S-ketamine. Antidepressants significantly increased LRRC54 and PgR expression, comparable to E2 and relative to vehicle control. Pretreatment with fulvestrant attenuated these effects, indicating ERα dependency. Data are presented as mean ± SEM from three independent biological replicates (n = 3). Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns as not significant.

Article Snippet: Imipramine hydrochloride (Sigma-Aldrich, I7379), fluoxetine hydrochloride (LKT Labs, F4780), and S-ketamine (Pfizer, 2750 Ballerup, Denmark) were used as antidepressants and 17-β estradiol (Sigma-Aldrich, E2758) served as the positive control in the study.

Techniques: Gene Expression, Phospho-proteomics, Western Blot, Activation Assay, Expressing, Control

Journal: bioRxiv

Article Title: Antidepressants interact with sex steroid receptors and their intracellular signaling components

doi: 10.64898/2026.03.17.712321

Figure Lengend Snippet: Time-dependent structural changes over 100 ns trajectories for ERα complexed with estradiol, imipramine, fluoxetine, S-ketamine, PaPE-1, and hydroxynorketamine (HNK). ( A ) Ligand root mean square deviation (RMSD) relative to the initial equilibrated structure, characterizing the stability and mobility of each compound within the binding pocket. ( B ) Protein backbone Cα RMSD, demonstrating the global structural integrity and stability of the receptor across all systems. ( C ) Cα root mean square fluctuation (RMSF) per residue, illustrating local protein flexibility and conserved dynamic patterns across the ligand-binding domain. ( D ) Radius of gyration (Rg) and its principal components (X, Y, Z), representing the maintenance of global structural compactness and shape. ( E ) Solvent accessible surface area (SASA) of the protein–ligand complexes, indicating stable exposure of the receptor-ligand surface throughout the simulation. ( F ) Visual representation of comparative binding free energies for the compounds based on MM/PBSA and MM/GBSA methods.

Article Snippet: Imipramine hydrochloride (Sigma-Aldrich, I7379), fluoxetine hydrochloride (LKT Labs, F4780), and S-ketamine (Pfizer, 2750 Ballerup, Denmark) were used as antidepressants and 17-β estradiol (Sigma-Aldrich, E2758) served as the positive control in the study.

Techniques: Binding Assay, Residue, Ligand Binding Assay, Solvent