Defects in microtubule dynamics in AKAP9-depleted ECs. (A) HUVECs treated with control or AKAP9 siRNA were fixed and subjected to immunofluorescence microscopy using antibodies to α-tubulin or EB1. A radial network of α-tubulin positive MTs (arrowhead), EB1 as comets (arrowhead), and microtubule organizing center (MTOC, open arrowhead) were present in control siRNA-treated cells. MTs formed a disorganized network (arrowhead) in AKAP9 siRNA cells. EB1 was visible at the MTOC (open arrowhead), but appeared as dot-like structures (arrowhead) in the cytoplasm. Total EB1 levels were comparable in control and AKAP9 siRNA-treated cells, as assessed by Western blotting of cell lysates. Actin served as a loading control. (B) Live-cell analysis of GFP-EB1–transduced control and AKAP9 siRNA-treated cells. Instantaneous microtubule growth rate was calculated from the displacements of EB1-positive microtubule ends between successive frames. The length of uninterrupted growth represents the entire length of the GFP-EB1 tracks. Each data point represents the growth rate or length of a single microtubule. The horizontal bar represents the median value for 80 MTs analyzed in 10 cells per group from 3 independent experiments. Distribution of growing ends along the cell radius is calculated as the number of EB1 comets present in successive quadrants (numbered 1-5) from the centrosome to the cell periphery. Fifteen cells were analyzed. Catastrophe frequency is a measure of the number of MTs that failed to reach the cell periphery (defined as a 3-μm zone from the cell boundary) in a given time. N = 80 MTs in n = 10 cells per group collected from 3 independent experiments. *P < .05. (C) Lysates were prepared from control and AKAP9 siRNA cells under basal conditions (Ba) and following nocodazole treatment and subsequent washout for the indicated times in minutes. The level of Glu-tubulin was evaluated in Western blots. α-Tubulin (tub) served as a protein loading control. One of 3 representative experiments is shown.