NOD-mCherry–induced syntelic kt-MT attachments are stable and exhibit reduced levels of Mad1. (A) Maximum projection of two-color confocal Z-sections from a NOD-mCherry–expressing cell with syntelic attachments (S1–S4) and bioriented chromosomes with amphitelic attachments (A1–A3). (B) Quantification of k-fiber fluorescence intensity of the amphitelic (A1–A3) and syntelic attachments (S1–S4) highlighted in A. The tubulin fluorescence presented in the bar graph is the integrated fluorescence intensity per micrometer of k-fiber. (C) Flow chart outlining the cold stability assay used to probe syntelic k-fiber stability. (D) Micrographs of no/low NOD-mCherry– and high NOD-mCherry–expressing control and cold-treated cells. Note that both syntelic (S) and bioriented (B) chromosomes retain their k-fibers equally after cold treatment. (E) Quantification of tubulin fluorescence intensity for no/low NOD-mCherry– and high NOD-mCherry–expressing cells for control and cold-treated cells. (F) Flow chart outlining the protocol used to examine Mad1 reduction at syntelic attachments. (G) Spinning disk confocal imaging of NOD-mCherry– and Mad1-YFP–expressing cells before and after a 15-min colchicine treatment to depolymerize the spindle microtubules. The fold increase (58× and 8×) in Mad1 levels after the colchicine treatment is shown for each cell. Error bars represent the SEM. Bars, 10 µm.