Figure 4.

Cyclin D Overexpression in hESCs Induces Neuroectoderm Differentiation

(A) Morphology of cyclin D overexpression (OE). Representative colonies of GFP OE and cyclin D OE hESCs.

(B and C) Cyclin D OE overexpression causes neuroectoderm differentiation and decreases endoderm markers. Expression of neuroectoderm markers in cyclin D overexpressing cells shown by qPCR (B) or western blot (C).

(D and E) Cyclin Ds repress endoderm loci. Luciferase constructs with Sox17 (D) or GSC (E) promoter regions containing Smad2/3 binding sites were cotransfected with cyclin D OE constructs, then differentiated into endoderm for 48 hr and analyzed for luciferase activity.

(F) Cyclin Ds repress the initiation of endoderm differentiation in early G1 phase. Fucci-hESCs transfected with cyclin D OE constructs were sorted into early G1 phase and analyzed for marker expression by flow cytometry after endoderm differentiation.

(G) Cyclin D knockdown causes the accumulation of Smad2/3 on chromatin. Relative amount of Smad2/3 protein in cytoplasm and on chromatin in cyclin D1-3 knockdown cells compared to Scramble shRNA overexpressing cells.

(H) Cyclin D overexpression results in Smad2/3 accumulation in the cytoplasm. Smad2/3 localization in cytoplasm and on chromatin was analyzed in cyclin D1, D2, and D3 overexpressing cells by western blot. All data are shown as mean ± SD. (n = 3). Student’s t test was performed. ^∗p < 0.05.

See also Figure S4.