Hec1-S55 phosphorylation in RPE-1 cells during metaphase is dependent on Aurora A residing at the spindle. (A) Schematic diagram of tagging endogenous TPX2 with mAID-mClover tag by the CRISPR/Cas–based method. (B) Depletion of mAID-mClover-TPX2 by IAA and expression of TPX2 constructs in RPE-1 cells arrested in metaphase. RPE-1 cells containing mAID-mClover-TPX2 and stably expressing indicated TPX2 constructs were treated as in Fig. S3 A, then harvested and lysed for immunoblot analysis using antibodies as indicated. Parental RPE-1 cells were used as a control. (C) Depletion of mAID-mClover-TPX2 by adding IAA to RPE-1 cells arrested in metaphase. Cells were treated as in Fig. S3 A, then fixed and stained with anti-GFP (mClover, green) and anti-TPX2 (red) antibodies. DNA was stained with DAPI (blue). Cells arrested in metaphase are shown. Scale bar: 5 µm. (D) Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase expressing an Aurora A mutant that cannot localize to the spindle. RPE-1 cells containing Aurora A–mAID-mClover and expressing mCherry–Aurora A (WT, S155R [SR], or kinase-dead [KD]) were treated as in Fig. S3 A, then fixed and stained with anti-Hec1 (blue) and anti–phospho-Hec1-S55 (green) antibodies. The mCherry signal (red) was acquired by fluorescence microscopy. Cells arrested in metaphase are shown. Boxed regions in the panels are shown as magnified images in insets. Scale bar: 5 µm (white), 500 nm (yellow). (E) Quantification of the Hec1-S55 phosphorylation in RPE-1 cells arrested in metaphase expressing Aurora A that cannot localize to the spindle. Relative intensity of the phospho-Hec1-S55 signal in cells treated as in D was calculated and displayed as in Fig. 2 B. The median of DMSO-treated cells was set as 1. The data represent a minimum of 105 kinetochores from five cells for each condition. P values were obtained using the Steel–Dwass multiple comparisons test. (F) Phosphorylation of Aurora A–T288 in RPE-1 cells expressing mCherry–Aurora A–WT, S155R, or K162R. Cells used in D were treated as in Fig. S3 A, fixed, and stained with anti–phospho–Aurora A–T288 (red) and anti-mCherry (green) antibodies. The mClover signal (blue) was acquired by fluorescence microscopy. Cells arrested in metaphase are shown. Scale bar: 5 µm. (G) Quantification of the Aurora A–T288 phosphorylation at spindle poles in RPE-1 cells expressing mCherry–Aurora A–WT, S155R, or K162R. Cells were treated as in F, and intensity of the phospho–Aurora A–T288 signal against mCherry–Aurora A signal is shown. Error bars represent SD of a minimum of five cells for each condition. P values were obtained using the Dunnett’s multiple comparisons test. (H) Aurora A distribution on the spindle in prometaphase (prometa) and metaphase. RPE-1 cells expressing Aurora A–mAID-mClover were fixed and stained with anti-GFP (mClover, green) and anti–α-tubulin (α-tub; red). DNA was stained with DAPI (blue). Scale bar: 5 µm. Intensities of Aurora A–mAID-mClover are plotted as in Fig. 3 F in the graphs on the right. BSD, blasticidin S deaminase; Dox, doxycycline; 3E, TPX2-F307E F334E F335E; N, N-terminal fragment (aa 1–43) of TPX2.