Figure 8. CDK5RAP2 promotes G2 arrest in... | Rockefeller University Press

Figure 8.

$Figure 8. CDK5RAP2 promotes G2 arrest in the presence of DNA damage. (A) Colony-forming ability of cells assayed 7 or 10 d after plating. n = 4; 40 cells per experiment. (B) Mitotic index was determined for wt and gene-disrupted DT40 cells that were incubated in DMSO for 10 h (+DMSO) or first irradiated with 20 Gy and then incubated in nocodazole for 10 h (+IR+NOC) or incubated in nocodazole for 10 h without irradiation (+NOC; bottom graph). n = 3; minimum of 3,000 cells was scored per condition per experiment for each genotype. P-values were calculated by two-tailed unpaired Student’s t test. (C) Levels of Chk1 protein are reduced in CNN1-deficient centrosomes. Protein fractions containing centrosomes purified from tagAKAP-wt or tagAKAP-cnn1lox cells (same purification as in Fig. 6 A) were immunoblotted with antibodies against centrin-1 and Chk1 kinase. Signal intensities of Chk1 in fractions 3 and 4 were normalized against the centrin signal in the same fraction. Table shows the percentage of Chk1 in tagAKAP-cnn1lox compared with tagAKAP-wt centrosomal fractions. WCE, whole cell extract. (D) 16 h treatment with aphidicolin (+APH) induces centrosome overduplication in DT40 cells. Caffeine alone (+CAF) or with aphidicolin (+APH+CAF) does not cause centrosome overduplication. Centrosome number was determined using γ-tubulin staining. Note that cnn1−/− cells contain slightly elevated centrosome numbers under all conditions. n = 3; at least 150 cells were scored per condition per experiment for each genotype. P-value was calculated by two-tailed unpaired Student’s t test. (E) Summary of the respective roles of the CNN1 and CNN2 domains in vertebrate cells. √, essential; ×, dispensable. Error bars represent SD.$

CDK5RAP2 promotes G2 arrest in the presence of DNA damage. (A) Colony-forming ability of cells assayed 7 or 10 d after plating. n = 4; 40 cells per experiment. (B) Mitotic index was determined for wt and gene-disrupted DT40 cells that were incubated in DMSO for 10 h (+DMSO) or first irradiated with 20 Gy and then incubated in nocodazole for 10 h (+IR+NOC) or incubated in nocodazole for 10 h without irradiation (+NOC; bottom graph). n = 3; minimum of 3,000 cells was scored per condition per experiment for each genotype. P-values were calculated by two-tailed unpaired Student’s t test. (C) Levels of Chk1 protein are reduced in CNN1-deficient centrosomes. Protein fractions containing centrosomes purified from tagAKAP-wt or tagAKAP-cnn1^lox cells (same purification as in Fig. 6 A) were immunoblotted with antibodies against centrin-1 and Chk1 kinase. Signal intensities of Chk1 in fractions 3 and 4 were normalized against the centrin signal in the same fraction. Table shows the percentage of Chk1 in tagAKAP-cnn1^lox compared with tagAKAP-wt centrosomal fractions. WCE, whole cell extract. (D) 16 h treatment with aphidicolin (+APH) induces centrosome overduplication in DT40 cells. Caffeine alone (+CAF) or with aphidicolin (+APH+CAF) does not cause centrosome overduplication. Centrosome number was determined using γ-tubulin staining. Note that cnn1^−/− cells contain slightly elevated centrosome numbers under all conditions. n = 3; at least 150 cells were scored per condition per experiment for each genotype. P-value was calculated by two-tailed unpaired Student’s t test. (E) Summary of the respective roles of the CNN1 and CNN2 domains in vertebrate cells. √, essential; ×, dispensable. Error bars represent SD.