Figure 4.

Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B and Cdk1. (A, C, and D) Normal, MCPH1^427insA, and PCNT^3109G>T LBCs were synchronized in G₁/early S phase by a mimosine block, released for 8 h to reach G₂ phase, and subsequently costained with rabbit anti–P-S230-Cdc25B (green) and mouse anti–γ-tubulin (red) antibodies (A), rabbit anti–P-Y15-Cdk1 (green) and mouse anti–γ-tubulin (red) antibodies (C), or mouse anti-Cdk1 (red) and rabbit anti–γ-tubulin (green) antibodies (D) and analyzed by confocal microscopy. (B) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdc25B. The mean percentages of cells with centrosomal colocalization of γ-tubulin and P-S230-Cdc25B are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: ***, P = 0.0006 (MCPH1^427insA); **, P = 0.006 (PCNT^3109G>T). (E) Loss of Chk1 from centrosomes induces activation of centrosome-associated Cdk1. The mean percentages of cells with centrosomal colocalization of γ-tubulin and total Cdk1 (light gray bars) or P-Y15-Cdk1 (dark gray bars) are indicated. Statistical significance versus control (LBC) by two-tailed Student's t test is as follows: *, P = 0.013 (MCPH1^427insA); P = 0.019 (PCNT^3109G>T). (F) Reduced levels of P-S230-Cdc25B and P-Y15-Cdk1 in isolated centrosome preparations from MCPH1^427insA and PCNT^3109G>T LBCs. Immunoblots were performed on three sucrose centrifugation fractions of centrosome preparations (left) and whole cell lysates (right) as an input control from control (LBC), MCPH1^427insA, and PCNT^3109G>T LBCs using antibodies against P-S230-Cdc25B, P-Y15-Cdk1, Cdk1, and, for comparison, Nek2 as a loading control and Mcm7 to exclude nuclear contamination. For whole cell lysates, antibodies to Cdc25B, Cdk1, and actin were included. Arrowheads point to centrosomes, which are shown enlarged in insets. Error bars represent the standard deviation after combining the results of three different experiments. Bars, 10 µm.