A) Clathrin heavy chain colocalizes with alpha-tubulin at the spindle apparatus in mitotic DKO-R cells.
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Confocal immunofluorescence microscopy on an asynchronised population of DKO-R cells grown in the absence of doxycycline (upper left panel). Cells were stained with Hoechst 33258 (blue; Sigma), anti-alpha-tubulin DM1A (green; AlexaFluor 488 donkey anti-mouse conjugate, Invitrogen) and anti-CHC Ab21679 (red; AlexaFluor 594 goat anti-rabbit conjugate, Invitrogen). The asterisk indicates a mitotic cell in which CHC is targeted to the kinetochore fibres. A Z-stack projection of the indicated mitotic cell is shown (right panel). Scale bar, 10 µm. B) Repression of CHC to undetectable levels is achieved after 48 hours treatment with doxycycline. Western blot analysis of DKO-R cells treated with doxycycline for the indicated time periods. β-tubulin was used as a loading control. C) Cell cycle distribution is unaffected by CHC depletion. DKO-R cells were treated with doxycycline for 48–72 hours and then subjected to flow cytometric analysis. DNA content was monitored by PI staining. The cell cycle profiles of CHC-expressing and CHC-depleted cells are shown. The percentage of cells in each cell cycle phase was calculated by ModFit Sofware and is shown in the boxed legend. D) The recovery kinetics from cell cycle arrest at the G2/M checkpoint are unaffected by CHC depletion in DKO-R cells. Time-course analysis of DKO-R cells following release from a nocodazole-induced block. Cells were grown in the presence (CHC-depleted) or absence (CHC-expressing) of doxycycline for 48–72 hours and then synchronized at metaphase by treatment with 500 ng/ml of nocodazole for 11 hours. A representative vehicle-treated asynchronous sample is indicated for each population (‘Asynch’ bar). At the indicated time points post-release from nocodazole block, cells were harvested and subjected to propidium iodide flow cytometric analysis. Data represent the mean values from three independent experiments with error bars denoting standard errors. The post-arrest recovery kinetics were compared for the clathrin-expressing and clathrin-depleted cells at each phase in the cell cycle using the analysis of variance (ANOVA). A p-value of 0.05 or less would be required to declare a significant difference between the two populations. P-values were 0.20 for G1-phase, 0.16 for S-phase and 0.09 for G2/M phase, indicating that there is no evidence of a difference between the recovery kinetics of the two populations over time following a nocodazole-induced block.