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Fig. 6. siRNA targeting of E-like leads to an increase in the stability of the microtubule cytoskeleton. (A) Depletion of E-like levels upon siRNA transfection. HeLa cells were transfected with either E-like siRNA (El) or a scrambled RNA-oligo duplex (Sc) and harvested at 48, 72 and 96 hours following transfection. Cell lysates were analyzed by immunoblotting using an E-like-specific antibody and a ß-actin antibody as a control for gel loading. (B) Reduced levels of E-like increase microtubule resistance to nocodazole and to Triton X-100 extraction. a-l, HeLa cells were transfected with either scrambled (Sc) or E-like (El) oligoduplexes and treated with nocodazole to completely depolymerize the microtubule cytoskeleton. At the end of the incubation, cells were restored to drug-free medium, fixed at the time points shown (0-15 minutes), and examined by immunofluorescence using anti--tubulin or anti-acetylated tubulin (acet-tub) antibodies. Inset shows an area of a cell at higher magnification to more clearly illustrate a population of intact microtubules. m,n, Control and E-like silenced cells were fixed after brief extraction with Triton X-100 and examined by immunofluorescence using an anti-acetylated tubulin antibody. (C) E-like siRNA does not modify the dynamics of single microtubules in EGFP--tubulin expressing HeLa cells (no siRNA: 16 cells, 59 MTs; with siRNA: 26 cells, 82 MTs). Bar, 10 µm. Vrs, velocity of rapid shortening; Ve, elongation velocity.

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