Fig 2: Cic represses Ets factor expression in the dendritic growth of hippocampal neurons. (A) Localization of Cic (green) and Etv4 (top) (red) or Etv5 (bottom) (red) in dissociated mouse hippocampal neurons after DIV13. Nuclear staining with DAPI is also shown. Last panel showed merged images. (B) Localization of Cic (green) and Etv4 (top) (red) or Etv5 (bottom) (red) in coronal sections of the mouse hippocampus. (C) Hippocampal culture lysates from DIV 1 to 21 and mouse hippocampal homogenates obtained at the indicated stages were immunoblotted to detect Cic, Etv4, and Etv5. a-Tubulin was used as a loading control. (D) HT22 cells were infected for 3 days with either lenti-control vector or lenti-Cic-OE, and WB was used to ascertain the change in Cic and Ets following overexpression of Cic in HT22 cells. (E) shows quantification of WB in d. (F–H) Hippocampal neurons cultured in vitro were infected on DIV 7 for 7 days with either lenti-scramble ShSCR or lenti-ShRNA against Cic (ShCic-1, ShCic-2). (F) mRNA levels of Ets were assessed by qPCR following Cic knockdown in hippocampal neurons. (G) The expression of Ets following Cic knockdown in neurons was detected by WB. (H) Quantification of the WB in G. The error bars indicate the SE. ***p < 0.001; **p < 0.01; *p < 0.05.

Fig 3: ERM binds to MED25. (A) Schematic summary of the interaction between ERM and MED25 proteins. The N-terminal TAD of ERM interacts with the ACID of MED25. NR: Nuclear receptor box. Numbers refer to amino acid. (B) Deletion analysis of ERM shows that the N-terminal 38–72 domain is sufficient for binding MED25 in vitro. GST fusion proteins of the indicated ERM fragments were used to assess the binding to full-length rabbit reticulocyte lysate in vitro generated Flag-MED25. Binding was detected by autoradiography (upper panel) or immunoblotting with anti-Flag (bottom panel). An SDS gel stained with Coomassie showing the expression of the GST fusion proteins is shown. (C) Deletion analysis of MED25 shows that the ACID domain is sufficient for binding to ERM 38–72 in vitro. GST and GST-ERM 38–72 were incubated with the indicated MED25 fragments produced in reticulocyte lysate. Binding was detected by immunoblotting with anti-Flag or anti-GFP. (D) Co-immunoprecipitation of MED25 with ERM. Flag-MED25 and wild-type or mutants ERM (upper panel) or mutants Flag-MED25 together with full-length ERM (bottom panel) were expressed in RK13 cells. Cellular extracts were immunoprecipitated with anti-Flag antibody (IP a Flag) and immunoblotted with anti-Flag (IB a Flag) and anti-ERM (IB a ERM) antibodies. Aliquots of the same extracts were analysed with the same antibodies to detect exogenous proteins (cellular extract). (E) Interaction between endogenous proteins. MDA-MB 231 nuclear extracts were subjected to immunoprecipitation with anti-Gal4 DBD (IP Ctrl) or anti-ERM (IP ERM) antibodies. Interactions were detected by western blot using polyclonal anti-MED25 (IB a MED25) or polyclonal anti-ERM (IB a ERM).

Fig 4: Expression of key genes involved in sex differentiation together with new potentially important genes (i.e. GLI1, ETV5, WNT2B) in male and female gonads aged 40–68 days pc. Red represents female, blue represent males.

Fig 5: Effects of MED25 on transcriptional activation by the ERM TAD. (A–D) U2OS cells were transfected with either Gal-ERM 1–72 (A, B and D) or full-length ERM (C) with (A and C) full length MED25, (B and C) MED25 ACID and MED25 VWA domains or (D) siRNA-resistant MED25 wild-type or derivatives. Cells collected 24 h after plasmid transfections were processed for luciferase activity. The relative luciferase activity of Gal-ERM 1–72 alone was assigned a transactivation level of 100%. Data represent the mean ± S.E.M. of at least three independent transfections performed in duplicate. (A) MED25 inhibits Gal4-ERM 1–72 transcriptional activation. Transient expression of MED25 in U2OS cells inhibits Gal4-ERM 1–72 transactivation but not activation by Gal4-E1A 13S or Gal4-E2F. (B) MED25 ACID and VWA domains inhibit Gal-ERM 1–72 transcriptional activation. (C) MED25 inhibits ERM transcriptional activity. (D) MED25 is required for full transcriptional activation by Gal4-ERM 1–72. siRNA-depleted cells were transfected with Gal-ERM 1–72 and reporters and analysed for luciferase activity. The expressed MED25 derivatives (WT, ?ACID, ?Ct, ?Nt, Q451E, M470A) were resistant to degradation induced by siRNA against wild-type MED25. The MED25 siRNA is directed against a sequence encoding the human ACID domain and therefore does not affect either the expression of MED25 ?ACID or the expression of MED25 WT, MED25 ?Ct, MED25 ?Nt, MED25 Q451E and MED25 M470A in which the human ACID domain has been replaced by its murine counterpart. (Inset) The levels of expression of MED25 after transient knockdown (as monitored by western blot with an antibody against MED25) and actin (loading control) are shown. The level of expression of the MED25 deletion mutants (western blot with anti-Flag antibody) after transfection into U2OS cells is also shown.