Smurf2 is a novel regulator of the spindle assembly checkpoint. (a) Smurf2 localizes at centromeres when the spindle checkpoint is active. HeLa cells were synchronized by a double-thymidine protocol (see Materials and methods). Cells were then released into a fresh medium (time 0), and 3 h later nocodazole was added. Immunofluorescence microscopy after staining with ACA, anti-Smurf2 antibody, and DAPI for DNA. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole. (b) Smurf2 depletion results in failed accumulation of APC/C-Cdc20 substrates during nocodazole-induced metaphase arrest. Immunoblotting was performed for mitotic regulators in HeLa cells transfected with Smurf2 siRNA #1 or nonspecific dsRNA (siNS). After release from the thymidine block, cells were incubated for the indicated hours in the presence of nocodazole. (c) Smurf2-depleted cells override nocodazole-induced spindle assembly checkpoint, resulting in tetraploidy. Control (siNS) and Smurf2-depleted cells were fixed at the indicated times and subjected to flow cytometry. (d) Cosilencing of Cdc20 restores mitotic accumulation of cyclin B1 and securin in Smurf2-depleted HeLa cells. Cells were synchronized and released into nocodazole-containing medium as described in b. (e and f) Smurf2 depletion results in loss of Mad2 from centromeres, whereas centromere localization of BubR1 is unaffected. Cells were fixed 8.5 h after release from the thymidine in the presence of nocodazole, and immunofluorescence microscopy was performed. (a, e, and f) The close-up images are shown with threefold higher magnification. Bars, 10 μm.