Loss of MISP leads to spindle misorientation. (A) HeLa cells treated with MISP or control siRNA for 48 h were stained for α-tubulin (green), γ-tubulin (red), and with Hoechst 33342 (blue). (B) Quantification of the described mitotic defects in control or MISP siRNA-treated cells. n = 50 cells each from three independent experiments. (C) Representative images of z-sections (0.4 µm per stack) with maximum spindle pole intensity and a side view of a 3D-reconstruction of the depicted cell with the spindle angle α. Cells were stained for α-tubulin (red), γ-tubulin (green), and Hoechst 33342 (blue). (D) Quantification of the percentage of metaphase cells with indicated spindle angles as described in C; n = 150 cells from three independent experiments. (E) HeLa cells treated with CENP-E, MISP, or control siRNA for 48 h were stained for α-tubulin (green), γ-tubulin (red), and with Hoechst 33342 (blue). Top, representative images with z-projection; middle, the x-z projection of γ-tubulin signal; bottom, scatter plots of the spindle angles in control cells, CENP-E, or MISP-depleted cells (n = 50 cells from three independent experiments) with calculated mean value. (F) Scatter plots showing the spindle angles in control, MISP siRNA-treated cells with and without expression of GFP or siRNA-resistant versions of GFP-MISP WT/6DP/7AP/9AC (n = 30 cells from two independent experiments) with calculated mean value. Bars: (A, C, and E) 5 µm. Error bars in B and D represent SD. Student’s t test was used to calculate p-values for comparison of control and experimental samples.