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. 2013 Jan 10;8(1):e53633. doi: 10.1371/journal.pone.0053633

Figure 2. Knockdown of 14-3-3ε by siRNA causes partial meiotic resumption mouse oocytes.

Figure 2

(A) 120 GV-oocytes microinjected with 14-3-3ε siRNA or control siRNA (5pl of 20 µM) were collected 24 h after microinjection. Oocytes microinjected with control siRNA and no injection served as control. mRNA of 14-3-3ε was detected by RT-PCR using primers specific for 14-3-3ε. (B) Protein expression of 14-3-3ε was examined by western blot with an anti-14-3-3ε antibody. β-actin used as endogenous internal reference demonstrate 14-3-3ε siRNA specificity and equal loading. (C) at the indicated times, the percentages of germinal vesicle breakdown (GVBD) or MII or death were counted in cultured mouse oocytes after 14-3-3ε siRNA or control siRNA microinjection. GVBD, 24 h; MII or death, 30 h. The total number of oocytes undergoing meiotic maturation or death is given on top of bar graph from three independent experiments. (D) H1 kinase activity in oocytes injected with 14-3-3ε siRNA or control siRNA or no injection groups. Each value was expressed as mean±S.D of at least three independent experiments. (E) Protein extracts from oocytes microinjected with 14-3-3ε siRNA or control siRNA and no injection groups were incubated with histone H1 and [γ-32P] ATP. The protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and incorporation of 32P into histone H1 was visualized by autoradiography. (F) Western analysis of phosphorylation status of Cdc2-Tyr15. GV oocytes injected with 14-3-3ε siRNA or control siRNA were collected 24 h after microinjection. The collected oocytes were immunoblotted with anti-pTyr15 of Cdc2 antibody. (G) Co-localization of Endogenous Cdc25B and 14-3-3ε in mouse oocytes injected with 14-3-3ε siRNA or control siRNA. red fluorescent Cdc25B signals and green fluorescent 14-3-3ε signals were co-localized in the cytoplasm in oocytes injected with control siRNA, while red fluorescent Cdc25B signals were translocated from the cytoplasm to the nucleus in oocytes injected with14-3-3ε siRNA. Scale bar = 20 µm.