Fig. 1.

Human importin-α1 is required for mitotic spindle assembly. (A) The expression level of importin-α1 is increased in mitosis. Cell extracts from asynchronous and nocodazole-arrested mitotic HeLa cells were prepared, subjected to SDS-PAGE and analyzed by immunoblotting with anti-importin-α1 and α-tubulin antibodies. The numbers below the bands refer to the relative gray value intensity. (B) HeLa cells were transfected with negative control (NC) siRNA or importin-α1 siRNA at 100 nM for 72 h. Cells were fixed and immunostained using antibodies against importin-α1, α-tubulin, KIFC1 and TPX2. DNA (blue) was stained using DAPI. Typical images are shown. (C) Monastrol-released MT regrowth was affected under importin-α1 knockdown. HeLa cells were treated with NC siRNA or importin-α1 siRNA for 72 h and were released into warm medium for 12 min and 28 min from monastrol-arrested mitosis and then were fixed and immunostained with anti-α-tubulin antibody. (D) Percentage of normal spindle formation 28 min after release from monastrol. Experiments were repeated three times, and at least 100 cells were measured for each group. Results are mean±s.d. (E) Importin-α knockdown results in a prolonged metaphase arrest in HeLa cells. HeLa cells were transfected with siRNA and RFP–H2B (as a chromatin marker, red) for 72 h and were subjected to automated time-lapse live-cell imaging. The onset of NEBD is marked as 0 min. (F) The average time from NEBD to sister chromosome separation of cells in E was calculated and statistically analyzed. Results are mean±s.d., n=50 cells per group. ***P<0.001 (Student's t-test). (G) Analysis of the efficiency of siRNA knockdown by immunoblotting using anti importin-α1 and GAPDH antibodies. Scale bars: 10 µm.