Fig. 2: Chk2 works in concert with JAK2 to promote PLK1 activity.
From: Chk2 sustains PLK1 activity in mitosis to ensure proper chromosome segregation
A Quantification of relative change in CFP/FRET ratio 1 h after addition of ATMi (10 μM KU55933) or DMSO in 100 ng/mL nocodazole-arrested U2OS cells. DMSO values are the same as in Fig. 1D. Each point denotes one cell. Values are combined from ≥3 experimental replicates. n = 56 cells for DMSO, n = 23 cells for ATMi. ns, not significant (p > 0.05), two-tailed t-test. Error bars SD of individual cell values. B Top, experimental setup. Following 4-h treatment with nocodazole (NOC, 100 ng/mL), mitotic cells were collected via shake off and centrifugation. These mitotic cells were then resuspended in a smaller volume of the original media and treated for 1 h with PLK1i (1 μM BI-2536), Chk2i (10 μM BML-277), ATMi (10 μM KU55933), DNA-PKi (5 μM M3814), ATRi (10 μM AZ-20), or equal volume DMSO. Following inhibitor treatment, cells were collected and immunoblotted. Right, quantification of ≥3 experimental replicates. Each point demarcates value from biological replicate. N = 5 DMSO, N = 5 PLK1i, N = 2 Chk2i, N = 3 ATMi, N = 4 DNA-PKi, N = 5 ATRi. Error bars SD. *p < 0.05, two-tailed t-test. ns, not significant. C Top, experimental setup. Isolated mitotic HeLa cells were treated for 1 h with PLK1i (1 μM BI-2536), Chk2i (10 μM BML-277), JAK2i (5 μM JAK2 inhibitor IV), combination of Chk2i and JAK2i, or equal volume DMSO as a vehicle control. Following inhibitor treatment, cells were collected and immunoblotted. Left, representative western blot. Right, quantification of 4 experimental replicates. Each point demarcates the value from one experimental replicate. Error bars SD. *p < 0.05, one-way Anova with Bonferroni multiple comparison correction. D Top, experimental setup. Isolated mitotic A549 WT and Chk2−/− cells were treated as in 2C. Left, representative western blot. Right, quantification of 4 experimental replicates. Each point demarcates value from biological replicate. Error bars SD. *p < 0.05, one-way anova with Bonferroni multiple comparison correction. All panels, data are presented as mean values ± SD. Source data are provided as a Source Data file.
