Fig. 5: Downregulating Mef2c can reverse for the enhanced neurogenesis in male offspring promoted by maternal hyperandrogen.

A Schematic of the IUE workflow and treatment timeline. At E14.5, vehicle- (Veh.) or letrozole- (LET) treated embryos were electroporated with an EYFP plasmid carrying either scramble shRNA (shCont) or Mef2c shRNA. B Cortical tissues were collected at E18.5 from male embryos for immunostaining (N = 4 males from 4 dams per group). Representative confocal images (left) and quantification of EYFP⁺ cell distribution across cortical layers (right) are shown. Scale bar, 50 μm. Green, red, and blue asterisks indicate comparisons between Veh. + shCont vs Veh. + shMef2c, Veh. + shCont vs LET + shCont, and LET + shCont vs LET + shMef2c, respectively. C–F Mouse primary NPCs were infected with shCont or shMef2c lentivirus and treated with DMSO or DHT (10 nM). Western blotting was performed to assess MAP2, GFAP, Nestin, and MEF2C levels (N = 4 per group). Vinculin served as the loading control. Representative blots are shown in (C), and quantification in (D–F). G Immunofluorescence staining of MAP2, GFAP, and Nestin in NPCs infected with EGFP-expressing shCont or shMef2c viruses. Representative images (left) and quantification of marker-positive cells among EGFP⁺ cells (right) are shown (N = 4 independent cultures). Scale bar, 20 μm. All data are expressed as the means ± SEM. Exact P-values are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Two-way ANOVA followed by Sidak post-hoc test was performed for (B). One-way ANOVA followed by Tukey post-hoc test was performed for (D–G).