ETAA1-dependent ATR activation regulates Aurora B kinase activity. (A) Diagram of mitotic ATR signaling to Aurora B (AURKB). (B and C) CHK1 pS317 (B) or Aurora B (C) pT232 levels were measured by imaging of mitotic cells. To avoid induction of replication stress, cells were arrested in G2 by the addition of the CDK1 inhibitor (RO-3306; 10 µM) for 16 h before addition of ATRi or IAA for 2 h and then release into fresh media containing taxol. Cells were fixed 1 h after release. ACAs were used to confirm kinetochore localization of Aurora B. Shown are representative immunofluorescent images and quantification of three independent experiments with at least 100 cells analyzed per condition. Error bars represent standard deviation. (D) pH3 S10 was measured in synchronized U2OS WT, ETAA1-null (ΔETAA1), and ΔETAA1 cells stably complemented with an ETAA1 expression vector. Shown are representative immunofluorescent images and quantification of three independent experiments with at least 100 cells analyzed per condition. Error bars represent standard deviation. Significance for B–D was determined by ANOVA with a Dunnett multiple comparison post-test. Bars: 5 µm (B–D).
