Stable HeLa Flp-In, hTERT-RPE1 Flp-In, and Bub1 mutant cell lines were constructed according to the manufacturer’s protocol (Invitrogen). We amplified the Bub1 cDNA by PCR and inserted it into the pcDNA5/FRT/V5-His-TOPO vector (Invitrogen). Flag-EGFP was subcloned C-terminally of Bub1. The mutants were constructed via site-directed mutagenesis. Cells were grown at 37°C in 5% CO2 in either Dulbecco’s minimum essential medium + 10% FCS (HeLa cells) or 50:50 Ham’s F-12/DME + 10% FCS (hTERT-RPE1 cells). HeLa Kyoto H2B-mRED cells (provided by D. Gerlich, ETH Zurich, Zurich, Switzerland) were supplemented with 500 µg/ml G418, HeLa Flp-In, and hTERT-RPE1 Flp-In cells with 400 µg/ml zeocin, and stable Bub1 mutant cells were supplemented with 300 µg/ml hygromycine (HeLa) or 5 µg/ml puromycine (RPE1). Cells were transfected as described with 30- (HeLa) or 40-nM (RPE1) siRNAs (Bub1 siRNA, 5′-GAGUGAUCACGAUUUCUAA-3′; alternative Bub1 siRNA, 5′-AAGATGCATTTGAAGCCCAGT-3′) and analyzed 48 h after transfection (Elbashir et al., 2001 (link)). Cells were treated for 1 h with 1 µM MG132 prior to fixation to measure congression efficiency. To measure spindle checkpoint activity, cells were treated with 1 nM nocodazole for 16 h, and the fraction of rounded-up cells was determined by phase-contrast microscopy. To measure apoptosis, cells were incubated with 1 nM nocodazole for 16 h and fixed with 3.7% formaldehyde in phosphate-buffered saline, pH 7.4. A TUNEL assay was performed using an in situ cell death detection system that contained fluorescein–deoxy UTP (dUTP; Roche).