Skip to main content
. Author manuscript; available in PMC: 2014 Mar 26.
Published in final edited form as: Oncogene. 2012 Nov 19;32(39):10.1038/onc.2012.501. doi: 10.1038/onc.2012.501

Figure 5. Ablation of TIF1γ expression by RNA interference delays progression through mitosis and activates the SAC.

Figure 5

(A and B) TIF1γ knockdown increases the number of cells with misaligned chromosomes at metaphase. HeLa cells were treated with either non-silencing siRNA or siRNAs specific for TIF1γ and seeded onto glass slides. 72h post-transfection cells were fixed and permeabilized and then co-stained with either α-tubulin, TIF1γ and DAPI, TIF1γ and DAPI, or Bub1, TIF1γ and DAPI, to gauge mitotic index, mitotic distribution and SAC activation. Nocodazole-treated cells were included as a control. (A) pro, prophase; prometa, prometaphase; meta, metaphase; ana, anaphase; telo, telophase. TIF1γ knockdown cells exhibiting a bipolar spindle and misaligned chromosomes at metaphase, were scored as metaphase cells. The bar chart represents results from three independent experiments (At least 300 mitotic cells were counted per experiment). Error bars denote standard deviation. *P<0.05; **P<0.01. (B) Immunofluorescent visualization of Bub1 associated with unattached kinetochores and attached kinetochores not under full tension in TIF1γ knockdown cells (276 out of 300 TIF1γ-1 metaphase knockdown cells had misaligned chromosomes and positive Bub1 staining whilst 253 out of 300 TIF1γ-2 metaphase knockdown cells had misaligned chromosomes and positive Bub1 staining compared to 17 out of 300 for non-silencing controls). (C) TIF1γ knockdown activates the SAC. HeLa cells were treated with either non-silencing siRNA, or siRNAs specific to TIF1γ. The levels of TIF1γ and the SAC proteins, BubR1 and Bub1 were determined by Western blotting 72h post knockdown. (D) BubR1 knockdown relieves SAC activation imposed by TIF1γ knockdown. HeLa cells were treated with either non-silencing siRNA, or siRNAs specific for TIF1γ BubR1, or TIF1γ and BubR1. Cell lysates were harvested after 72h and levels ofTIF1γ, BubR1, phospho Ser10-histone H3 (pH3), histone H3 (H3), cyclin B1 and β-actin were all determined by Western blotting. (Ei) TIF1γ associates with the APC/C-MCC in SAC-activated cells. HeLa cells were treated with nocodazole (200 ng/ml) for 20h. Cell lysates were then prepared and APC7 and TIF1γ were immunoprecipitated. The relative amounts of TIF1γ, APC7, Cdc20 and BubR1 associated with APC7 and TIF1γ were determined by Western blot. APC7 (s), short exposure; APC7 (l), long exposure. (Eii) HeLa cells were treated with either non-silencing siRNA, or siRNAs specific for TIF1γ APC7 and TIF1γ were immunoprecipitated from cell lysates prepared 72h post-transfection. The relative amount of BubR1 associated with APC7 was determined by Western blot. (F) HeLa cells were treated with either non-silencing siRNA, or siRNAs specific for TIF1γ or APC15 and 48h later, treated with nocodazole (200 ng/ml) for 20h. The association of Cdc20, BubR1 and Bub3 with the APC/C was assessed by Western blot following immunoprecipitation with an anti-APC4 antibody.