Figure 2.

Cited2 Is Important for Early Steps of Cardiac Differentiation

(A) Timeline depicting the protocol used for differentiation of mouse ESC from D0 onward. The time of ethanol or 4HT treatment and the days of beating activity assessment are indicated.

(B) Expression of Cited2 determined by qPCR at D0, D2, D3, D5, and D12 of differentiation in cells derived from C2^fl/fl[Cre] ESC treated at D0 with 4HT or ethanol for 48 hr.

(C) CITED2 protein levels determined by western blotting in extracts from C2^fl/fl[Cre] ESC, 48 hr after incubation with ethanol or 4HT. Loading in each lane was monitored by detection of β-TUBULIN.

(D) Percentage of colonies with beating foci derived from C2^fl/fl[Cre] ESC treated with ethanol or 4HT at D0, D2, D4, and D6 of differentiation.

(E) Quantification by flow cytometry of cells expressing cTNT at D12, treated as described in (B). The percentage of cells expressing cTNT over the total number of cells is indicated.

(F) Expression levels determined by qPCR of Myh6, cTnT, Foxa2, α-fetoprotein (Afp), Nestin, and βIII-tubulin (βIII-tub.) at D12 of differentiation in cells treated as described in (B).

(G) Immunofluorescent detection of α-ACTININ (left panels, red staining) and MYOSIN HEAVY CHAIN MF20 (right panels, red staining) in C2^fl/fl[Cre] ESC-derived cells at D10 of differentiation, after treatment for 2 days with ethanol (upper panels) or 4HT (lower panels) at D0. Nuclei were counterstained using DAPI (blue), and cells were examined on at 100× magnification.

Results in (B), (D), (E), and (F) are presented as the mean ± SEM of three independent biological experiments.