MARK2 regulates mitotic microtubule growth rate and function. (a) Single plane deconvolved images of cells immunostained with antibodies against anti–β-tubulin after control or MARK2-1 siRNA treatment. Bars, 5 µm. Grayscale image signals are inverted to highlight astral microtubule length and density. (b) Graph comparing the distribution of instantaneous velocities of EB3 comets in cells expressing EB3-mKate and treated with either MARK2 or control siRNA. Values were obtained manually by clicking spots using SoftWoRx software. Nonoverlapping peak values between control and MARK2 siRNA–treated cells signify statistically significant differences (*, P < 0.01) using the proportion test. Error bars are SEM values across cells from two independent repeats. (c) Bar graph shows frequency distribution of spindle polarity (based on the number of spindle poles: one [mono], two [bi], three to four, or more than five). Cells were treated with siRNA as indicated, exposed to 1.7 µM nocodazole for 3 h to depolymerize all microtubules, recovered for 10 min in nocodazole-free medium to reassemble spindles, and immunostained using α-tubulin antibody. 100 nM 2ME2 was added in the recovery medium of control siRNA–treated cells. Error bars represent SEM from three independent experiments.