Figure 1. Dido3 Is Necessary for Early Embryonic Stem Cell Development.

(A) The three isoforms encoded by the Dido3 locus (Dido1, Dido2, and Dido3).

(B) Expression of Dido3 was analyzed in Wt/Wt embryos at E7.5 by immunofluorescence.

(C) Expression of Dido3 and Oct4 was analyzed by western blot in 3T3 and primary MEF somatic fibroblasts and in pluripotent embryonic and induced stem cells; β-actin was used as loading control.

(D) Wt/Wt and ΔCT/ΔCT ESCs were aggregated into EBs and maintained without LIF in untreated bacterial plates for the time indicated to induce differentiation. In western blot, we analyzed Oct4 and endogenous Dido3 expression in WT ESCs and their EBs, as well as Oct4 and Dido3ΔCT in mutant ESCs and EBs.

(E) In contrast to Dido3, which shows no significant differences (data not shown), Dido1 is upregulated at the onset of differentiation in Wt/Wt cells but not in ΔCT/ΔCT cells. Quantitative RT-PCR was used to determine Dido1 expression; values were normalized to actin.

(F) ΔCT/ΔCT ESCs expressing HA-DidoNT were treated as in (D) and analyzed by western blot for expression of Oct4, endogenous Dido3ΔCT, as well as for overexpressed HA-DidoNT.

(G) Quantitative RT-PCR was used to determine expression of selected stemness markers in d10 ΔCT/ΔCT EBs (blue bars) overexpressing HA-DidoNT (green) or HA-DidoPHDm (red).

Data show mean ± SD (n = 3) for one representative experiment of two performed, indicating expression of the markers analyzed in cells transfected with HA-DidoNT or HA-DidoPHDm, relative to d10 Wt/Wt EBs (baseline).

See also Figure S1.