Figure 2

Loss of 14-3-3ε results in block in G2/M transition. A: 150 fertilized eggs microinjected with14-3-3ε siRNA or control siRNA (10 pl of 20 μmol) were collected 15 h after microinjection. RT-PCR is used for detection of mRNAs of 14-3-3ε (800 bp) and β-actin (300 bp). B: Western blot analysis of 14-3-3ε protein expression at 15 h after microinjection of 14-3-3ε siRNA using anti-14-3-3ε or anti-β-actin antibodies (upper panel). 300 fertilized eggs are loaded onto each lane. Densitometric quanitification represents the protein expression of 14-3-3ε (lower panel). A x² test was used to evaluate the differences of endogenous 14-3-3ε expression between multiple experimental groups. Bars represent means ± S.D of three independent experiments. *P <0.01 vs. no injection or control siRNA group. C: The cleavage rate in cultured mouse embryos of 14-3-3ε siRNA, control siRNA and no injections at 31 h after hCG injection. The cleavage rates were calculated with data from three independent experiments. The total number of eggs undergoing division, dead or abnormal cleavage is given on top of the bar graph. A x² test was used to evaluate the differences between multiple experimental groups. Bars represent means ± S.D of three independent experiments. ^*P <0.01, ^△P <0.05, ^▲P > 0.05 vs. no injection or control siRNA group. D: Morphology changes of mouse fertilized eggs in 14-3-3ε siRNA microinjection and control groups. Representative images are shown. Bar =50 um. a) Eggs in no injection group. b) Eggs in control siRNA group. c) Eggs in 14-3-3ε siRNA group. d) Abnormal cleavage in 14-3-3ε siRNA group. The arrows indicate the eggs with abnormal cleavage. E, F: MPF activity was detected at indicated time points in 14-3-3ε siRNA and control groups. For each point, eggs were lysed and MPF activity was examined by scintillation counting (E) and autoradiography (F). One-way analysis of variance followed by a Least Significant Difference (LSD) test was used to evaluate the differences in the MPF activity assay. Bars represent means ± S.D of three independent experiments. G: Western blot analysis of phosphorylation status of CDC2-Tyr15. The eggs were collected at indicated time points after 14-3-3ε siRNA injection in all the examined groups. 200 eggs are loaded onto each lane.