Fig. 6

Increasing the relative level of soluble (non-MT) tubulin promotes HSET-driven aster formation in cells. a Generation of a transgenic HeLa cell line where EGFP-HSET expression is under control of doxycycline. Western blot of whole cell lysates probed with antibodies that recognize EGFP, tubulin, and HSET. Numbers indicate days of induction with doxycycline/DMSO. Green arrow indicates overexpressed EGFP-HSET, and black arrow represents endogenous HSET. b Maximum intensity z-projections of metaphase EGFP-HSET HeLa cells treated with DMSO (top) or doxycycline (bottom) for 3 d, then fixed and stained with antibodies against tubulin (yellow), centrin (magenta) or Hoechst (DNA, blue). Numbered boxes correspond to spectrally unmixed centrin-stained regions shown on right. Y/N indicates the presence of centrin at spindle poles. Scale bar, 5 µm. c EGFP-HSET HeLa cells were pretreated with DMSO (top) or doxycycline (bottom) for 3 d, then treated with 500 nM nocodazole (Noc) for 15 min to increase the relative levels of soluble tubulin. Cells were fixed, stained, and imaged identically to b and maximum intensity z-projections are shown. Scale bar, 5 µm. d Quantification of b, c. After analyzing the tubulin/centrin channels, the fraction of cells containing ≥1 acentrosomal aster was compared between 3 d doxycycline/DMSO cells after treatment with 500 nM nocodazole for 0, 15, and 30 min. Overlaid dots represent the average observed fraction for each independent experiment. ^***p < 0.001 by two-tailed t-test. Data were reported as the average ± SEM of N = 3 independent experiments with n ≥ 60 cells for each condition. e EGFP-HSET HeLa cells were treated with doxycycline for 3 days to induce maximal expression, and 500 nM nocodazole was added to increase relative levels of soluble tubulin. Maximum intensity z-projections were acquired in the EGFP channel every 3 min and a representative time-course is shown for the indicated times. Scale bar, 5 µm