FIGURE 9.

MeCP2 deficiency affects microtubule nucleation. A, MRC-5 cells were transfected with siMeCP2#1 or #2 and their respective control (Ctr.) siRNAs, and, after 4 days of silencing, microtubules were depolymerized by adding 10 μg/ml nocodazole for 1 h. Microtubule regrowth was tested by releasing the cells in fresh medium for 2 or 5 min, and then they were fixed and stained with anti α-tubulin (red). Nuclei were stained with DAPI (blue). The silencing of MeCP2 was verified by Western blotting, using actin as loading control (n = 3 for each siRNA). Scale bars = 10 μm. B, microtubule nucleation capacity of the centrosome in cells silenced or not silenced for MeCP2 in one representative experiment of three independent experiments showing the same tendency. After nocodazole treatment, the cells were released for 2, 5, and 10 min (n = 300 cells). C, aster area in cells after 5-min release upon nocodazole treatment (n = 70 cells in three independent experiments, mean ± S.E.). ***, p < 0.001; unpaired Student's t test.