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. 2022 Jun 6;25(7):104536. doi: 10.1016/j.isci.2022.104536

Figure 4.

Figure 4

ATR-T1989p localization is independent of PIK and CHK1 kinase activities but dependent upon CDC7

(A) Localization of ATR-T1989p to the midbody of late cytokinetic cells upon ATR inhibition (ETP-46464, 10 μM; 1 h).

(B) Quantification of ATR-T1989p intensity at the midbody of late cytokinetic cells upon ATR inhibition (ATRi-1 = ETP-46464, ATRi-2 = VE-821; 1 h) across three independent experiments (ns, Student’s t-test).

(C) Localization of ATR-T1989p to the midbody of late cytokinetic cells upon ATM (KU55933, 10 μM; 1 h) or DNA-PK (NU7441,10 μM; 1 h) inhibition.

(D) Quantification of ATR-T1989p intensity at the midbody of late cytokinetic cells upon ATM (KU55933, 10 μM; 1 h) or DNA-PK (NU7026,10 μM; 1 h) inhibition across three independent experiments (ns, Student’s t-test).

(E) Localization of Aurora B-T323p to the midbody of late cytokinetic cells upon CHK1 inhibition (MK-8776, 10 μM; 1 h).

(F) Quantification of Aurora B-T323p intensity at the midbody of late cytokinetic cells upon CHK1 (MK-8776, 10 μM; 1 h) inhibition across three independent experiments (∗p = 0.0162, Student’s t-test).

(G) Localization of ATR-T1989p to the midbody of late cytokinetic cells upon CHK1 inhibition (MK-8776, 10 μM; 1 h).

(H) Quantification of ATR-T1989p intensity at the midbody of late cytokinetic cells upon CHK1 inhibition (MK-8776, 10 μM; 1 h) across three independent experiments (ns, Student’s t-test).

(I) Localization of CHK1 to the midbody of late cytokinetic cells upon ATR inhibition (ETP46464, 10 μM; 1 h).

(J) Quantification of CHK1 intensity at the midbody of late cytokinetic cells upon ATR inhibition (ETP46464, 10 μM; 1 h) across three independent experiments (ns, Student’s t-test).

(K) Localization of ATR-T1989p to the midbody of late cytokinetic cells upon CDC7 inhibition (CDC7i-1 = XL413, 10 μM, CDC7i-2 = PHA-767491, 10 μM; 1 h).

(L) Quantification of ATR-T1989p intensity at the midbody of late cytokinetic cells upon CDC7 inhibition (CDC7i-1 = XL413, 10 μM; CDC7i-2 = PHA-767491, 10 μM; 1 h) across three independent experiments (∗p = 0.0165, ∗∗p = 0.0388; Student’s t-test).

(M) Percentage of cells in cytokinesis upon ATRi (ETP-46464, 10 μM; 1 h) and/or CDC7i (XL413, 10 μM; 1 h). At least, 400 cells were analyzed in each of 3 independent experiments (∗∗p = 0.0015, 0.0063, and 0.0016 for ATRi, CDC7i, and ATRi/CDC7i, respectively; Student’s t-test). All dot plots represent data from three independent biological replicates, each replicate is represented by a different symbol. All data have been normalized to the control sample. Data are represented as mean ± SD of the individual replicates. HeLa cells were used in A–M. See also Figure S4.