Figure 1.
Rbbp4 is essential in the maintenance of self-renewal and pluripotency in ESCs
(A) Schematic representation of the production for conditional inactivation of Rbbp4 in ESCs.
(B) Western blot showing RBBP4 levels in Rbbp4F/F-transfected Cre recombinase for different time points.
(C) Morphology of ESC colonies of indicated genotypes. Bright-field images of ESC colonies after 7 days of culture (grown from a single ESC). All ESC colony images were photographed at day 7 after seeding single-cell suspensions on feeder layers. Scale bar, 100 μm. The outlines of obviously defective colonies were circled in red. Rbbp4Δ/Δ ESCs expressing empty FLAG vector (Rbbp4Δ/Δ/EV) and vectors encoding wild-type RBBP4-FLAG (Rbbp4Δ/Δ/WT) are also shown.
(D) Secondary ES colony-replating assay. Bar graph shows the number of cells with AP-positive staining in the absence of RBBP4. Error bars represent means and STD from three 6 cm dishes.
(E) ESC growth competition assay. Oct4 knockout ESCs serve as a negative control.
(F) Representative fluorescence-activated cell sorting plots of annexin V and propidium iodide (PI) levels in Rbbp4F/F and Rbbp4Δ/Δ ESCs. Percentages of cells with different apoptosis marker levels are shown.
(G) Cell-cycle analysis of indicated ESCs. Percentages of cells in different phases are indicated. Representative histograms presented here show distribution of cells in sequential phases (G0/G1, red; S, light blue; and G2/M, red) of cell cycle.
(H) Alkaline phosphatase activity staining on each indicated ESC colony. Scale bar, 100 μm.
(I) Western blot analysis of OCT4, SOX2, NANOG, RBBP4, and RBBP7 protein expression in indicated ESCs; ACTIN served as a loading control.
See also Figures S1 and S2.