Depletion of TRAMM results in a mitotic arrest as a result of a chromosome congression failure. (A) HeLa cells were photographed by brightfield microscopy at 20× magnification 24 h after treatment with siRNA against the TRAPP subunit indicated or Mad2. In some cases, the cells were cotransfected with siRNA-resistant or -sensitive TRAMM. For simplicity “Trapp” is omitted for each subunit. Depletion was assessed by Golgi morphology using anti–mannosidase II and Western analysis for subunits to which antibodies were available (Fig. S1 A). Bars: (brightfield images) 200 µm; (fluorescence images) 5 µm. (B) Cells from A were quantitated by counting the number of mitotic cells in multiple fields over multiple experiments. Error bars indicate SD. Significance was assessed by an unpaired t test. (C) HeLa cells were fixed 24 h after treatment with nonspecific (NS; top row) or TRAMM/TrappC12-specific (bottom row) siRNA. Staining was performed with antitubulin, ACA, and 7-aminoactinomycin D. Bars, 5 µm. (D) HeLa cells expressing fluorescently labeled histone H2B were treated with NS (top row) or TRAMM/TrappC12-specific (bottom row) siRNA. The cells shown were recorded 20 h after treatment. Frames from Videos 1 and 2 were captured, and times indicated are relative to nuclear envelope breakdown (NEBD). Bars, 10 µm. (E–G) Measurements of chromosome boundary dimensions (E), interkinetochore distances (F), and unaligned chromosomes (G) were made after treatment of HeLa cells as in A. Chromosome boundary length (L) and width (W) were measured between the spindle poles and perpendicular to the poles, respectively. Results are shown ± SD, and significance was assessed by an unpaired t test. KD, knockdown.