MT attachment stability was restored by inhibiting Aurora B kinase or CLASP^Mast/Orbit overexpression. (A) Monopolar spindles were induced after double kinesin-5^Klp61F/kinesin-8^Klp67A RNAi and treated with 20 µM Binucleine-2, an inhibitor of Drosophila Aurora B kinase, or control DMSO. (B) Frequency of chromosome flipping (per cell, per second; ± SEM) in Binucleine-2–treated cells (n = 7 [control DMSO treated] and 8 cells [Binucleine-2 treated]). (C) GFP-CLASP^Mast/Orbit overexpression in the monopolar spindle depleted of kinesin-5^Klp61F and kinesin-8^Klp67A. (D) A representative chromosome (inset in C). Chromosomes were static when GFP-CLASP^Mast/Orbit was overexpressed. (E) Frequency of chromosome flipping (per chromosome, per second; ± SEM). Expression of GFP-CLASP^Mast/Orbit significantly reduced the frequency of flipped chromosomes (*, P < 0.034; **, P < 0.0013; Games-Howell test). RNAi (and overexpression) were performed two or more times, and combined data are presented. Each dot in the graph represents mean frequency for a cell. A total of 168 chromosomes in 15 cells (control), 364 chromosomes in 24 cells (kinesin-8^Klp67A RNAi), 128 chromosomes in 13 cells (augmin^Dgt6 RNAi), and 128 chromosomes in 11 cells (kinesin-8^Klp67A RNAi and GFP-CLASP^Mast/Orbit overexpression) were analyzed. Bars, 5 µm.