Figure 3.

Cyclin Ds Are Necessary for Pluripotency

(A) Cell-cycle-dependent binding of Smad2/3 to endoderm genes. ChIP analyses in Tra-1-60+ sorted FUCCI-hESCs showing Smad2/3 binding on endoderm genes.

(B) Cyclin D expression during early differentiation of hESCs. Cyclin D1-3 protein expression during days 1–3 of neuroectoderm, endoderm, and mesoderm differentiation shown by western blot analysis.

(C) Morphology of cyclin D double knockdown. Representative colonies of shRNA Scramble and cyclin D double-knockdown cells.

(D) Triple knockdown of cyclin D causes endoderm differentiation. cyclin D1/3 double-knockdown cells were transfected with a cyclin D2 shRNA construct expressing GFP and then FACS sorted for qPCR analyses.

(E) Triple knockdown of cyclin D causes loss of pluripotency markers. Immunofluorescence microscopy analyses for Oct4, Nanog, and Sox2 expression (red) in cyclin D triple-knockdown hESCs (green / arrows).

(F) Double knockdown of cyclin D causes endoderm differentiation and blocks neuroectoderm differentiation. Cyclin D double-knockdown cells were analyzed for germ layer marker expression by western blot.

(G) Triple knockdown of cyclin D causes endoderm differentiation. Cyclin D1/3 double-knockdown cells were transfected with a cyclin D2 shRNA construct expressing GFP and then FACS sorted for western blot analyses. UD, undifferentiated cells. Student’s t test was performed. ^∗p < 0.05. Scale bar represents 100 μm.

See also Figures S2 and S3.