The long and short SKAP isoforms display distinct mitotic functionality. (A) Schematic of SKAP depletion and replacement protocol. Also see Fig. S2 A for a Western blot analyzing the efficiency of SKAP depletion and replacement. (B) IF images showing tubulin and DNA localization for the phenotypes exhibited during SKAP depletion and rescue experiments. From top to bottom: Aligned, multipolar spindles, misaligned (off-axis) chromosomes, multipolar spindles, and aligned chromosomes. Images represent maximal-intensity projections. (C) Quantification of mitotic chromosome alignment for SKAP depletion and rescue experiments from B. Cells were quantified by observing DNA and spindle morphology by IF. Cells were quantified for a phenotype if they displayed GFP reporter expression (if applicable) and displayed chromosome alignment and spindle and cell shape morphology indicative of metaphase or a multipolar mitotic cell morphology. (D) IF images showing a control cell with a bipolar spindle and paired centrioles and a SKAP-depleted multipolar cell with prematurely separated centrioles. (E) Quantification of mitotic timing (nuclear envelope breakdown [NEBD]-anaphase) as assessed from time-lapse videos at 40 h after siRNA addition (see Materials and methods). NEBD and anaphase were scored using DNA and cell morphology. Cells were counted if they entered mitosis during the course of filming. Cells that arrested for longer than 3 h or reached a terminal mitotic phenotype are boxed at the top of the graph. n = 52 cells per condition. Note: this analysis excludes cells in the depletion and long SKAP experiments that were arrested at the start of the video, and thus likely underrepresents the extent of the mitotic arrest. (F) Live cell imaging showing GFP-tagged SKAP cell lines for the two SKAP isoforms in interphase and mitosis. Bars, 5 µm.