MISP stabilizes astral MTs. (A) MISP or control siRNA-treated HeLa cells were stained with α-tubulin (green) and Hoechst 33342 (blue). (B) Schematic overview and quantification of the observed metaphase spindles. The image is the control cell shown in A. The intensities of the spindle (I_spindle) and the total cell (I_total) were determined with ImageJ software. Relative astral MT intensity (I_{astral, rel}) was calculated by [(I_total − I_spindle)/I_spindle] and control value was set to 1. n = 20 cells each from three independent experiments. (C) RFP-tubulin HeLa stable cells were transfected with MISP or control siRNA and stained for EB1 (green) and Hoechst 33342 (blue). Enlargements of the insets show EB1 localization in the cortical region (dashed lines). Right panel shows quantification of the percentage of metaphase cells with EB1-MT at the cortex; n = 50 cells each from three independent experiments. (D) Co-staining of MISP (red), α-tubulin (green), and Hoechst 33342 (blue) in fixed HeLa cells. Inset shows higher magnification of the framed region. (E) HeLa cells transfected with control or MISP siRNA, or MISP siRNA under expression of GFP or siRNA-resistant versions of GFP-MISP WT/6DP/7AP/9AC were stained for α-tubulin. Top, representative images with z-projection; bottom, quantification of percentage of metaphase cells with astral MTs. Bars: (A, C–E) 10 µm; (C and D, insets) 2.5 µm. Error bars represent SD. Student’s t test was used to calculate p-value for comparison of control and experimental samples.