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. 2014 May 30;20(9):850–860. doi: 10.1093/molehr/gau040

Figure 3.

Figure 3

CBP-CITED4 plays a role upstream of C/EBPβ during ovulation. (A) CBP inhibition impairs ovulation in vivo. WT mice (PD23) were super-ovulated by PMSG and hCG treatment. In the experimental group (n = 6), C646 was co-injected with hCG. Ovaries were paraffin-embedded and HE stained at 8 and 16 h post-hCG. Scale bar = 100 µM. (B) Rates of COC expansion in pre-ovulatory follicles at 8 h after hCG or hCG/C646 treatment. *P < 0.001. (C) Numbers of ovulated oocytes harvested from oviducts were counted at 16 h after hCG and C646 treatment. *P < 0.001. (D) Forskolin/PMA-induced CITED4 and C/EBPα/β expression in cultured GCs. Primary GCs were harvested from mice at PD23, cultured overnight and then treated with FSK (10 µM)/PMA (20 nM), U0126 (10 µM) or H89 (10 µM) for the indicated periods of time. Endogenous CITED4, C/EBPα and β expressions after the indicated treatments were detected by western blotting. (E) The upper panel showed the Luciferase assay results for the effects of forskolin/PMA and C646 (4 µM) on C/EBPβ activity in cultured GCs. GCs were transfected with a 2xC/EBP Luciferase reporter plasmid at 24 h before forskolin/PMA treatment, and Luciferase activity was assessed at 4 h after forskolin/PMA treatment. The lower panels showed the expression levels of endogenous CITED4 and C/EBPβ in the same GCs used for Luciferase assay. ERK1/2 was blotted as a protein loading control. *P < 0.001 when compared with sample 2 from left. (F) qRT–PCR results for relative Cited4 mRNA expression levels in GCs isolated from ovaries of WT and Cebpa/bgc−/−mice at 0, 4 and 24 h after hCG treatment. (G) Interaction of CBP-CITED4 with C/EBPβ in vivo. Lysates were prepared for the ovaries from mice at PD23 with or without hCG treatment for 4 h. Endogenous CBP was immunoprecipitated using an anti-CBP antibody. Co-precipitation of CITED4 and C/EBPβ was detected by western blotting. (H) Interaction of C/EBPβ with CITED4 in vivo. Lysates were prepared for the ovaries from PD23 mice at 4 h after hCG treatment. Endogenous C/EBPβ was immunoprecipitated using an anti-C/EBPβ antibody. Co-precipitation of CITED4 was detected by western blotting. As a negative control, the anti-C/EBPβ antibody was replaced with normal rabbit IgG in the parallel IP experiment.