(A and B) C2C12 myoblasts 48 hr after induction of differentiation treated with shRNA as indicated and stained for Myogenin (a marker for differentiating myoblasts, yellow), PCM-1 (red) and DAPI (blue). GFP-Tubulin (green) indicates successful transfection with shRNA. Insets show higher magnification of microtubule arrangement. Scale bars 25 μm, 5 μm in insets. (C) Timecourse of expression of embryonic myosin, a marker of myogenic differentiation. (D) Timecourse of relocalisation of PCM-1 from a focus around the centrosome to the surface of the nucleus. Cells with a complete nuclear ring were scored as positive. Data in C, D show mean ± SD, n = 30–50 cells from 2 experiments. (E) RNAi rescue experiment of delayed PCM-1 relocalisation phenotype. Data show mean ± SEM of 3 experiments with 50–60 cells each. (F) Timecourse of accumulation of cells with acetylated tubulin during muscle cell differentiation. Data in show mean ± SD, n = 30–50 cells from 2 experiments. (G) Manual tracing of microtubule filaments (yellow) relative to the longitudinal cell axis (red) in elongated mono-nucleated cells selected after 48 hr differentiation and shRNA treatment as indicated. Scale bars 10 μm. See supplementary Videos 6, 7. (H) Microtubule directionally from data as in g shown as cummulative frequency distribution. Data show mean ± SD, n = 3 experiments, 1522–1958 MTs. (I) Automatic tracking of EB3-tdTomato comets in elongated mono-nucleated cells selected after 48 hr differentiation and shRNA treatment as indicated. Direct lines from start to end of track are shown and colour-coded for direction relative to longitudinal axis of cell as indicated in legend. Scale bars 10 μm. See supplementary Videos 8, 9. (J) Distribution of microtubule growth angles obtained from data as in H. Data show mean ± SD, n = 3 experiments, 6251–6382 tracks. (K and L) Microtubule growth speed and duration was determined from EB tracks as in I. Pooled data shown as statistical box plots with percentiles as indicated.