PMC

(A) APC/C ligase activity directed towards cyclin A and cyclin B1 is reduced following TIF1γ knockdown. HeLa cells were treated with either non-silencing siRNA, or siRNAs specific for TIF1γ. The APC/C holoenzyme was immunoprecipitated from cell lysates 72h post-treatment using an antibody to APC3. APC/C activity was then assayed by incubating anti-APC3 immunoprecipitates with L-α-[³⁵S]-methionine labeled cyclin A or cyclin B1. After 1 h incubation samples were separated by SDS-PAGE and polyubiquitylation of cyclin A and cyclin B1 was assessed following autoradiography of the dried gel. (B) TIF1γ associates with cyclin A but not cyclin B1. HeLa cells were subject to immunoprecipitation with TIF1γ, and Western blotted for cyclin A or cyclin B1; GST and GST-cyclin A were incubated with L-α-[³⁵S]-methionine labelled TIF1γ GST- fusion, and binding proteins were purified upon glutathione-agarose beads and separated by SDS-PAGE. The binding of TIF1γ to GST-cyclin A was assessed following autoradiography of the dried gel. * indicates full-length GST fusion protein; upper band in GST lane is GST dimer. (C) Cyclin A is inappropriately present at metaphase in TIF1γ knockdown cells. 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 (i) or cyclin A and DAPI (ii)

(A) Mitotic APC/C substrates are stabilized following TIF1γ knockdown. HeLa cells were treated with either non-silencing siRNA, or siRNAs specific for TIF1γ. Cell lysates were prepared at the times indicated and levels of APC/C substrates determined by Western blotting. TIF1γ levels were determined to monitor knockdown efficiency and α-actin levels were determined as a loading control. (B) TIF1γ knockdown increases the mitotic index. HeLa cells were treated with either non-silencing siRNA, or siRNAs specific for TIF1γ Cells were fixed in 70% (v/v) ice-cold ethanol 72h post-transfection and subsequently subjected to FACS analysis, following co-staining with propidium iodide, to gauge cell cycle distribution, and an anti-phospho Ser10-Histone H3 antibody to gauge the numbers of cells specifically in mitosis. ***P<0.001; data taken from three independent experiments. Error bars represent standard deviation. (C) TIF1γ knockdown does not affect APC/C subunit expression. HeLa cells were treated with either non-silencing siRNA, or siRNAs specific for TIF1γ Cdc20 and Cdh1. Forty-Eight h post-knockdown cells were harvested and subject to SDS-PAGE and Western blotting.

(A) TIF1γ binds the APC/C preferentially in mitosis and S phase. HeLa whole cell extracts were prepared from G1, S, G2 and M phase enriched cell populations. APC7 was then immunoprecipitated from each fraction, isolated upon Protein G Sepharose, and the relative amount of TIF1γ associated with APC7 immune complexes was determined by Western blot. Cell cycle status was verified by Western blotting for cyclins A, B, phospho Ser10-Histone H3 (pH3) and α-tubulin. (B) The ability of TIF1γ to bind the APC/C decreases once the SAC is satisfied and cells progress through mitosis. HeLa cells were treated with 200ng/ml nocodazole for 20h. Mitotic cells were then isolated by shake-off and either harvested, or released back into cycle following the removal of nocodazole and then harvested at later times. APC7 was then immunoprecipitated from cell lystaes, isolated upon Protein G Sepharose and TIF1γ binding to the APC/C was assessed by Western blot. Levels of BubR1, cyclin B1, APC4, APC7, TIF1γ and α-tubulin were also assessed by Western blot. (C) TIF1γ binds Cdc20 preferentially in mitosis. HeLa whole cell extracts were prepared from G1, S, G2 and M phase enriched cell populations. Cdc20 and TIF1γ was then immunoprecipitated from each fraction, isolated upon Protein G Sepharose, and the relative amount of TIF1γ associated with Cdc20 was determined by Western blot. (D) Cdc20 knockdown does not affect APC/C association with TIF1γ HeLa cells were treated with non-silencing siRNA, Cdc20, and Cdh1 siRNAs. Forty-Eight h post-knockdown cells were harvested and subject to immunoprecipitation with anti-TIF1γ antibodies Following SDS-PAGE TIF1γ association with the APC/C was assessed by Western blotting.

HeLa cells expressing Histone H2B mCherry and α-tubulin-EGFP were treated with either an siRNA specific for luciferase or siRNAs specific for TIF1γ 48h post-knockdown cells were subjected to a thymidine block for 16 h, whereupon cells were released back into cycle and video imaging began 9 h later. Video images representative of treatments with (A) luciferase siRNA (B) TIF1γ-1i siRNA and (C) TIF1γ-2i siRNA are presented. Corresponding bar charts illustrating the time taken for cells to progress from NEBD to anaphase are also presented. ^ represents cells that failed to exit mitosis during filming. * represents cells that failed to undergo successful metaphase-to-anaphase transition during filming (depicted in Fig. 4E). DIC and immunofluorescent imaging revealed that those cells that failed to undergo metaphase-to-anaphase transition were characterized at late times by plasma membrane blebbing, compacted chromatin and aberrant mitotic spindle formation. Video imaging was therefore terminated at this time, and the time taken from NEBD to this point was recorded. The mean values calculated for time taken from NEBD to anaphase are therefore, underestimated for those cells that failed to undergo metaphase-to-anaphase transition. (D) Western blot for TIF1γ and α-tubulin in cells treated with luciferase siRNA, TIF1γ-1i or TIF1γ-2i siRNAs.

HeLa cells expressing Histone H2B mCherry and α-tubulin-EGFP were treated with either an siRNA specific for luciferase (A) or siRNAs specific for TIF1γ (B), BubR1 (C) or TIF1γ and BubR1 (D). 48h post-knockdown cells were subjected to a thymidine block for 16 h, whereupon cells were released back into cycle and video imaging began 9 h later. Bar charts depicting time taken to pass from NEBD to anaphase are presented. * represents cells that failed to undergo successful metaphase-to-anaphase transition during filming (depicted graphically in Fig. 7F). (E) Western blots for TIF1γ BubR1 and α-tubulin in cells treated with luciferase, TIF1γ BubR1, or γ and BubR1 siRNAs. (G) Expression of a siRNA-resisitant TIF1γ species allows for cyclin A degradation and mitotic progression. HeLa cells were treated with either non-silencing siRNA, or siRNAs specific for TIF1γ for 48 h. Cells were then transfected with vector alone or a construct expressing FLAG-TIF1γ and cultured for a further 24h. Cell lysates were prepared, and the levels of FLAG-TIF1γ, endogenous TIF1γ, cyclin A, phospho Ser10-histone H3 (pH3) and β-actin were all determined by Western blot.

(A-D) APC/C components, Cdc20, Cdh1 and TIF1γ were immunoprecipitated from asynchronous whole cell HeLa extracts, separated by SDS-PAGE, and Western blotted for coprecipitating proteins. APC7 (s), short exposure; APC7 (l), long exposure. (E) TIF1γ binds to APC3 and Cdc20 in vitro. GST and GST-APC2, APC3, APC5, APC6, APC7 and Cdc20 fusion proteins were incubated with L-α-[³⁵S]-methionine labeled TIF1γ GST-fusion, and binding proteins were purified upon glutathione-agarose beads and separated by SDS-PAGE. The binding of TIF1γ to GST proteins was assessed following autoradiography of the dried gel. *indicates full-length GST fusion protein; upper band in GST lane is GST dimer (F) APC/C complexes were immunoprecipitaed from asynchronous whole cell HeLa extracts using an anti-APC3 monoclonal antibody. APC/C immune complexes were assayed for E3 ligase activity in the presence of L-α-[³⁵S]-methionine labeled cyclin B1 or TIF1γ. After 1 h incubation samples were separated by SDS-PAGE. Polyubiquitylation of cyclin B1 and TIF1γ was assessed following autoradiography of the dried gel. (G) TIF1γ is post-translationally modified in response to nocodazole treatment but it is not degraded following nocodazole release (noc rel). HeLa cells were treated with 200ng/ml nocodazole for 20h. Mitotic cells were then isolated by shake-off and either harvested, or released back into cycle following the removal of nocodazole and then harvested at later times. Proteins were separated by SDS-PAGE and then Western blotted for TIF1γ, cyclin B1 and β-actin. Lane 1: asynchronous cells (A); lane 2: mitotic shake-off following nocodazole treatment (0); lanes 3-5: 1, 2 and 4h post nocodazole release. (H) TIF1γ is not targeted for degradation by Cdc20 or Cdh1. HeLa cells were transfected with mammalian expression plasmids expressing either Myc-tag alone (Myc-EV), Myc-tagged Cdc20, or Myc-tagged Cdh1. Samples were harvested 24h post-transfection and subject to SDS-PAGE and Western blotting. (I) Emi1 knockdown does not promote TIF1γ degradation. HeLa cells were treated with non-silencing siRNA, Emi1, and Cdh1 siRNAs. Forty-Eight h post-knockdown cells were treated with 200 ng/ml nocodazole for 20h. Mitotic shake-off cells were then released from the nocodazole block (noc rel) and harvested at the times indicated and subject to SDS-PAGE and Western blotting.

(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.