Fig. 4

YFP-PTIP stable line for mitotic process. A RNAi resistant YFP-PTIP was induced by doxycycline from HeLa Tet-on stable line and PTIP siRNA oligos were transfected for 24 h. Total lysates of stable line were resolved in SDS-PAGE and processed for western blot using PTIP antibody and GAPDH antibody. B HeLa Tet-on control or YFP-PTIP stable cell line was treated with PTIP RNAi for 18 h and subjected to live-cell imaging for 20 h. About 50 mitotic cells were examined for mitotic time. Red dots indicate cells that died in or just after mitosis. P-value was calculated by student t-test, paired, two-tailed. C HeLa Tet-on cells were first arrested at G1 by thymidine and transfected with PTIP siRNA oligos. Then, they are released from G1 block for 10 h and arrested at metaphase by MG132 for 1 h. Representative images of normal alignment or mild/severe misalignment were shown. Normal cells mean metaphase cells with all aligned chromosomes at the metaphase plate. Mild cells mean metaphase cells with the majority of chromosome aligned at the metaphase plate but the minority of chromosomes proximally located away from the metaphase plate. Severe cells are similar to the mild cells except misaligned chromosomes farther away from the metaphase plate. Size bar, 5 µm. D Statistics of panel C was shown. About 100 mitotic cells were counted in three replicates. P-value was calculated by student t-test, paired two-tailed