SSX2IP depletion does not interfere with centrosome-free spindle formation. (A) Expression of SSX2IP during in vitro maturation was specifically suppressed by MO oligonucleotides but did not alter meiotic spindle morphology; immunoblotting and indirect immunofluorescence detect SSX2IP and tubulin. (B and D) Chromatin-induced spindle assembly was monitored in X. laevis egg extracts that were mock-depleted (control, ct), immunodepleted of SSX2IP (ΔSSX2IP) or TPX2 (ΔTPX2), or supplemented with control (ctrl.) or anti (α)-SSX2IP antibodies (B). In D, control (ctrl.) antibodies or antibodies against Dynein intermediate chain were used. The blot in B shows depletion efficiencies; images display chromatin bead spindles (red, tubulin; green, SSX2IP; blue, DAPI). Please note that SSX2IP antibody addition efficiently inhibits pole localization of SSX2IP. (C) Quantification of assembled chromatin bead spindles in depleted extracts as indicated (left graph; this experiment was completed once, n = 100), or after addition of specific anti-SSX2IP or anti-TPX2 antibodies (right graph shows mean values ± SD of three independent experiments). The significance was calculated by a Student’s t test (two-tailed) and scored as follows: *, P < 0.05; **, P < 0.01. The magnified panels (magn.) show enlarged views of the boxed regions. Bars: (main panels) 20 µm; (magnified panels) 2 µm.