Figure 6.

Impaired microtubule regrowth from centrosomes in mitotic U2OS cells depleted of Hice1 and/or Hec1 by siRNA. (A) Immunostaining of α-tubulin and γ-tubulin in the mitotic U2OS cells subjected to microtubule regrowth assay. Cells were stained against α-tubulin and γ-tubulin. Confocal microscopy and image deconvolution were performed, and the maximal projections of z-axis series of images were shown. Luciferase siRNA-treated cells were analyzed as a control. Bar, 10 μm. (B) Time-dependent kinetics of centrosomal microtubule aster formation. Microtubule asters (0.5–3 min) were quantified by measuring the fluorescence intensity in a 4-μm circular area around each distinct centrosome. The mean value at each time point was derived from ≥30 randomly selected mitotic cells with a near bipolar spindle (three independent experiments) and used to fit a growth curve according to a multifactor exponential equation (SigmaPlot, Systat Software, San Jose, CA). The 12-min time point was not measured as the asters had become less distinct. AU, arbitrary units. (C) p values derived from a two-way ANOVA test (factor 1, time; factor 2, siRNAs) to show differences in the mean levels of microtubule aster intensity between various siRNA (factor 2) treatment groups. Differences among different time points (factor 1) are all significant (p < 0.002, data not shown).