Figure 1.
Silencing Geminin or Emi1 Mimics Depletion of the Pluripotency Factor Oct4 in P19 Mouse Embryonal Carcinoma Cells
(A) Geminin, Oct4, and Emi1 were depleted by siRNA in P19 embryonal carcinoma (EC) cells, and whole-cell lysates were harvested for analysis by western blotting. β-actin was used as a loading control. In mouse cells, a cross-reacting band lies below the Emi1 band marked with an asterisk.
(B) Geminin depletion by siRNA in P19 EC cells results in upregulation of trophectoderm markers Troma-1 and P-cadherin (both in green) and nuclear enlargement at 2 days and 6 days posttransfection. Total DNA is shown in red. Scale bars represent 20 μm.
(C) Trophectoderm markers Troma-1, P-cadherin, and Cdx2 (in green) are induced in P19 EC cells that lack geminin (purple). The mean proportion of cells lacking geminin was 67.8% of cells at 24 hr versus 43% at 48 hr as a result of outgrowth of untransfected cells (data not shown).
(D) Bar chart showing lineage analysis of control, geminin-, Oct4-, and Emi1-depleted P19 EC cells. The proportion of cells labeled by immunofluorescence with markers of extraembryonic (Troma-1, P-cadherin, Cdx2) and embryonic (β-III tubulin, Sox1, Gata6, and Brachyury) differentiation is shown. Eomes is a marker of both mesoderm and trophoblast differentiation. At least 1000 cells were scored, and all cells in a given field were scored. Error bars represent 5% standard error.
(E) Upregulation of trophectoderm markers is observed in P19 EC cells depleted of Oct4 at 2 days and 6 days posttransfection.
(F) Emi1 inhibits APC/CCdh1 activity to drive progression through the mitotic cell cycle by inhibiting the degradation of geminin and cyclins A2 and B1. Emi1 inactivation (or depletion) results in unopposed APC/CCdh1 activity and ubiquitin-mediated proteasomal destruction of its substrates geminin and cyclins.
(G) Emi1 depletion phenocopies depletion of geminin in P19 EC cells with upregulation of trophectoderm markers and gross nuclear enlargement observed at 2 days and 6 days. Total DNA is shown in red. Scale bars represent 20 μm.
For additional related data, see Figure S1.