Generation of Mps1 conditional–null and analogue-sensitive human cells. (A) Schematic of AAV vectors used to mutate the MPS1 locus. Circles and triangles denote FRT and loxP sites, respectively. ITR, inverted terminal repeat. (B) MPS1^flox/Δ cells were infected with the indicated adenoviruses and analyzed for Mps1 expression by immunoblotting. (C) MPS1^flox/Δ and MPS1^Δ/Δ cells complemented by wild-type (wt) or analogue-sensitive (as) Mps1 transgenes were analyzed by immunoblotting. LAP, localization and affinity purification. (D) Allele-specific inhibition of Mps1^as. Cells were cultured in the presence of the bulky purine analogue 3MB-PP1 (from left to right: 0, 0.078, 0.313, 1.25, and 5 µM) for 7 d and stained with crystal violet. (E) Cells were fixed and stained with antibodies to centromere autoantigens (CREST; red) and transgene-encoded Mps1^wt and Mps1^as (green). Bar, 10 µm. (F) Mps1^as inhibition overrides the SAC. Cells were filmed at 10-min intervals during treatment with nocodazole in the presence or absence of 10 µM 3MB-PP1 to assess the length of M phase (defined as the period of cell rounding by phase-contrast microscopy). Error bars indicate SEM.