p150CAF-1 Depletion and Loss of Heterochromatin Organization Are Compatible with Active DNA Replication in ES Cells
(A) ES cells were transfected with p150CAF-1 or control siRNA vectors. After 3 d under puromycin selection, cells were pulse-labeled for 10 min with BrdU and immediately analyzed by IF. No significant difference in BrdU incorporation could be detected between control and p150CAF-1-depleted cells. The right-hand image shows the merge between the p150CAF-1 and BrdU fluorescence. Scale bar = 10 μm.
(B) Immunodetection of PCNA (red) revealed no significant difference in the formation of replication foci between control and p150CAF-1-depleted ES cells. Immunodetection of p150CAF-1 is shown in green and the merging of p150CAF-1 and PCNA appears in yellow.
(C) Flow cytometry analysis showed a similar cell-cycle profile for control and p150CAF-1-depleted ES cells. Results are presented as the percentage of cells in each phase of the cell cycle (G1, S, G2/M), as defined by BrdU incorporation and DNA content. Data presented are the mean of three independent experiments; error bars indicate the standard deviation. All experiments were performed 3 d after transfection of the siRNA vectors.