Figure 1: Monopolar HEC1-9A cells satisfy the SAC.

(a) Cartoons illustrating the experimental set-up that uncouples stable attachments from biorientation. Cells expressing HEC1 variants and forced to be monopolar, as a consequence of treatment with STLC, will have various states of kinetochore–microtubule attachments, as listed below drawings. (b,c) Immunofluorescent labelling (b) and quantification (c) of indicated proteins in STLC-arrested cells. Cells were treated with MG132 for 2 h before fixation. Quantification is normalized to the kinetochore (KT) intensity of CENP-C and is the average fold change of three experiments (±s.e.m.) normalized to the values of control cells. Each dot represents one cell. The data points of individual experiments (n=49) are depicted in different shades of grey. Quantifications were subjected to unpaired Student’s t-test against the values measured for control cells. *P<0.01. (d) Quantification of the number of MAD2-positive kinetochores as a percentage of the total kinetochores per cell. Conditions and representation as in (b). (e) Time-lapse analysis of mitotic arrest in STLC-treated cells. Shown is the average of three experiments (solid lines)±s.e.m. (transparent area). (f,g) Time-lapse single-cell analysis (f) and representative stills (g) of HEC1-WT and HEC1-9A monopolar cells. For each cell, the total GFP-kinetochore level was measured at mitotic entry, normalized against the average level measured in HEC1-WT cells and plotted in (f). Filming started ∼1 h after release from CDK1-inhibitor RO-3306. 21 HEC1-WT, and 33 HEC1-9A cells were followed in two independent experiments. (h,i) Representative images (h) and quantification (i) of the number of MAD2-positive kinetochores in HEC1-WT and -9 A monopolar cells in time. Cells entered mitosis in the presence of STLC and subsequently treated with MG132 for the duration of the indicated time. Data are the average of two experiments (n=65). Arrowheads in h indicate MAD2-positive kinetochores in HEC1-9A cells that point away from the centrosome and move to the periphery or out of the chromatin pack. (j) Representative stills from live analysis of HEC1-WT and HEC1-9A-expressing RFP-MAD2. Arrowheads indicate MAD2-positive kinetochores. Filming was started 1 h after release from RO. Scale bars, 10 μm. Scale bars in other panels, 5 μm.