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. 2016 Oct 4;2:16033. doi: 10.1038/celldisc.2016.33

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Copyright © 2016 The Author(s)

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ASK1 activity is required for spindle orientation/positioning and astral microtubule stability. (a–c) Immunofluorescence images (a), spindle length (b) and spindle angle (c) of metaphase HeLa cells transfected with mCherry (red) in the absence or presence of ASK1^KD and stained with anti-α-tubulin (green) and anti-γ-tubulin (purple) antibodies and DAPI (blue). The position of the z stage is indicated in micrometers; 3D, xy projection. n=25 cells per group. Scale bars, 5 μm. (d, e) Immunofluorescence/phase-contrast images (d) and spindle displacement distance (e) of metaphase HeLa cells transfected as in (a) and stained with anti-α-tubulin antibody (green) and DAPI (blue). Red, mCherry. n=25 cells per group. Scale bars, 5 μm. (f) Immunoblots showing the levels of ASK1 phosphorylated at threonine 845 (pT845-ASK1), ASK1 and α-tubulin in HeLa cells treated with 0, 15 or 30 μm NQDI-1. (g–i) Immunofluorescence images (g), spindle length (h) and spindle angle (i) of metaphase HeLa cells treated with NQDI-1, and stained as in (a). n=25 cells per group. Scale bars, 5 μm. (j, k) Immunofluorescence/phase-contrast images (j) and spindle displacement distance (k) of metaphase HeLa cells treated with NQDI-1 and stained as in (d). n=25 cells per group. Scale bars, 5 μm. (l, m) Immunofluorescence images (l) and astral microtubule intensity (m) of metaphase HeLa cells transfected as in (a) and stained with anti-α-tubulin antibody (green) and DAPI (blue). Red, mCherry. n=30 cells per group. Scale bars, 5 μm. (n, o) Immunofluorescence images (n) and astral microtubule intensity (o) of metaphase HeLa cells treated with NQDI-1 and stained with anti-α-tubulin antibody. n=25 cells per group. Scale bars, 5 μm. (p, q) Immunofluorescence images (p) and astral microtubule intensity (q) of metaphase HeLa cells transfected with control or ASK1 siRNAs, treated with control (DMSO) or 0.5 μm paclitaxel, and stained with anti-α-tubulin antibody. n=25 cells per group. Scale bars, 5 μm. Experiments were performed three times. Values are mean±s.e.m. **P<0.01, ***P<0.001; NS, not significant.