T H E J O U R N A L O F C E L L B I O L O G Y

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1 T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Espert et al., Figure S1. Bub1 and BubR1 kinetochore localization are regulated by Mps1 and a PP2A-family phosphatase. (A) HeLa cells were spindle checkpoint arrested with nocodazole overnight, and then treated for the indicated number of minutes with an Mps1 inhibitor AZ3146/MG132 mix. The cells were then fixed and stained with BubR1 and Bub1 antibodies and CREST antiserum. (B) HeLa cells were control depleted or depleted of PP1 and - simultaneously (sippp1ca/c) for 84 h, including a 12-h nocodazole arrest. The cells were then either treated with MG132 (Control) or a MG132/5-iodotubercidine mix (Haspin inhibition) for 10 min followed by fixation and staining with antibodies to histone H3 phospho-thr3 (H3pT3). DNA was stained with DAPI. (C) HeLa cells were control depleted, depleted of PP1 and - (sippp1ca/c) as in B, or depleted of all PP2A-B56 subunits, nocodazole arrested, and then treated with Mps1 inhibitor AZ3146/MG132 mix for 5 min followed by fixation and staining with antibodies against BubR1 and CREST serum. Images are representative of three independent experiments. Bars, 10 µm. S1

2 Figure S2. PP2A-B56 controls BubR1 kinetochore localization and mitotic progression. (A) SAC-arrested HeLa cells depleted of individual PP2A-B56 subunits were treated with MG132 alone or a combination of MG132 and Mps1 inhibitor AZ3146 for 5 min, fixed, and stained with antibodies against BubR1 and CREST. (B) Normalized BubR1 kinetochore signal after AZ3146 addition was quantitated and plotted (mean ± SD [error bars]; n 120 kinetochores per bar). (C) Depletion efficiency of different PP2A-B56 subunits was assessed by Western blotting. (D) Depletion of PP2A-B56 results in mitotic arrest. HeLa S3 cells stably expressing GFP-tagged -tubulin and mcherry-tagged histone H2B (Zeng et al., 2010) were control or PP2A-B56 depleted and imaged on a spinning disc confocal microscope as they progressed through mitosis (nuclear envelope breakdown = 0 min). Image stacks were taken every minute. Representative maximum projections are shown. Bar, 10 µm. S2

3 Figure S3. PP2A-B56 dephosphorylates phospho-knl1 and is not dependent on Sgo2. (A) pknl1-pt875 signal depends on Knl1. HeLa cells were control depleted or Knl1 depleted for 48 h and then synchronized by a thymidine block. After release into fresh medium, cells were incubated for 7 h, then nocodazole and MG132 were added for an additional 3 h. Cells were fixed and stained for total Knl1 and pknl1-pt875. CREST serum was used to label kinetochores. (B) pknl1-pt875 signal depends on Mps1 activity. SAC-arrested HeLa cells were treated for 5 min with MG132 (control) or an MG132/AZ3146 mix (Mps1 inhibitor). The cells were fixed and stained with antibodies against Knl1-pT875, total Knl1, and CREST serum. (C) PP2A-B56 dephosphorylates phospho-knl1. GFP-Mis12 immunoprecipitates were incubated with control, PP2A-B56, or PP2A-B55 complexes as in Fig. 3 G. Inputs, beads, and supernatants were blotted as indicated. (D) Sgo2 depletion results in loss of PP2A-B56 from the centromere. HeLa cells were control or Sgo2 depleted for 48 h, arrested overnight with nocodazole, and fixed and stained for Sgo2 and PP2A-B56. Images are representative of three independent experiments. (E) Cell extracts of cells used in C were used for Western blotting with antibodies against Sgo2. Tubulin was used as a loading control. (F) HeLa cells were control or Sgo2 depleted for 48 h and arrested with nocodazole overnight. The cells were then treated with MG132 alone or MG132/ AZ3146 for 5 min, fixed, and stained with antibodies against BubR1 and Sgo2 and CREST serum. Bars, 10 µm. S3

4 Table S1. Phosphatase SmartPool sirna sequences Gene name Gene ID Accession number Target sequence (5 to 3 ) PPP2CA 5515 NM_ GAACUUGACGAUACUCUAA PPP2CA 5515 NM_ GCUUGUAGCUCUUAAGGUU PPP2CA 5515 NM_ GGCAAGAUAUUUCUGAGAC PPP2CA 5515 NM_ GCAAAUCACCAGAUACAAA PPP2CB 5516 NM_ CACGAAAGCCGACAAAUUA PPP2CB 5516 NM_ UUUAGUAGAUGGACAGAUA PPP2CB 5516 NM_ CCAGAACGCAUUACAAUAU PPP2CB 5516 NM_ GAACCAGGCUGCUAUCAUG PPP2R1A 5518 NM_ AGGCGGAACUUCGACAGUA PPP2R1A 5518 NM_ AAACUUAACUCCUUGUGCA PPP2R1A 5518 NM_ GUUCACAGAGCUCCAGAAA PPP2R1A 5518 NM_ GAGCUUCUGCCUUUCCUUA PPP2R2A 5520 NM_ CAUACCAGGUGCAUGAAUA PPP2R2A 5520 NM_ GUAUAGAGAUCCUACUACA PPP2R2A 5520 NM_ GCAAGUGGCAAGCGAAAGA PPP2R2A 5520 NM_ AGACAUAACCCUAGAAGCA PPP2R2B 5521 NM_ UCGAUUACCUGAAGAGUUU PPP2R2B 5521 NM_ GGGUCGGGUUGUAAUAUUU PPP2R2B 5521 NM_ GAAUGCAGCUUACUUUCUU PPP2R2B 5521 NM_ CCACACGGGAGAAUUACUA PPP2R2C 5522 NM_ CGGAGGAUCUUUGCCAAUG PPP2R2C 5522 NM_ GAUACAACCUGAAGGAUGA PPP2R2C 5522 NM_ CCAACAACCUGUACAUCUU PPP2R2C 5522 NM_ GAAGAUUACCGAACGAGAU PPP2R2D NM_ GUAGGUCCUUCUUCUCAGA PPP2R2D NM_ UCGGAUAGCGCCAUCAUGA PPP2R2D NM_ GAGACUACCUGUCGGUGAA PPP2R2D NM_ GAGAACGACUGCAUCUUUG PPP2R5A 5525 NM_ GCUCAAAGAUGCCACUUCA PPP2R5A 5525 NM_ CAAUACAAGUGCCGAAUAA PPP2R5A 5525 NM_ UGAAUGAACUGGUUGAGUA PPP2R5A 5525 NM_ GGAAAUGAAUGGCAAGCUU PPP2R5B 5526 NM_ CGCAUGAUCUCAGUGAAUA PPP2R5B 5526 NM_ UCAAGUCGCUGUCUGUCUU PPP2R5B 5526 NM_ CAAACCAUCGUAUCACUGA PPP2R5B 5526 NM_ GAACAAUGAGUAUAUCCUA PPP2R5C 5527 NM_ GGAUUUGCCUUACCACUAA PPP2R5C 5527 NM_ GGAAGAUGAACCAACGUUA PPP2R5C 5527 NM_ CAUCAGAAUUUGUGAAGAU PPP2R5C 5527 NM_ CAGAAGUAGUCCAUAUGUU PPP2R5D 5528 NM_ GUACAUCGACCAGAAGUUU PPP2R5D 5528 NM_ UCCAUGGACUGAUCUAUAA PPP2R5D 5528 NM_ UGACUGAGCCGGUAAUUGU PPP2R5D 5528 NM_ GUAGGCAGAUCAACCACAU PPP2R5E 5529 NM_ UUAAUGAACUGGUGGACUA PPP2R5E 5529 NM_ GCACAGCUGGCAUAUUGUA PPP2R5E 5529 NM_ GACACGCUAUCUGAUCUUA PPP2R5E 5529 NM_ GGAUAAAGUAGACGGAUUU PPP1CA 5499 NM_ GCAAGAGACGCUACAACAU PPP1CA 5499 NM_ GAGCAGAUUCGGCGGAUCA PPP1CA 5499 NM_ CAUCUAUGGUUUCUACGAU PPP1CA 5499 NM_ GAACGACCGUGGCGUCUCU PPP1CB 5500 NM_ GCAAAGUUAUGGUCGAUUU PPP1CB 5500 NM_ GCUGAGCGAUUUCUAAUAA PPP1CB 5500 NM_ GUGCUUAACUGUCUAAUAU PPP1CB 5500 NM_ GAGAAGAGACUUAAUCCAA PPP1CC 5501 NM_ GCGGAGAGUUUGACAAUGC PPP1CC 5501 NM_ CGAAUUAUGCGACCAACUG S4

5 Table S1. Phosphatase SmartPool sirna sequences (Continued) Gene name Gene ID Accession number Target sequence (5 to 3 ) PPP1CC 5501 NM_ UAGAUAAACUCAACAUCGA PPP1CC 5501 NM_ UGACAUCCAUGGACAAUAC PPP6C 5537 NM_ CUAAAUGGCCUGAUCGUAU PPP6C 5537 NM_ CGCUAGACCUGGACAAGUA PPP6C 5537 NM_ GUUUGGAGACCUUCACUUA PPP6C 5537 NM_ CGAACGGAAUCAGGAAAUU PPP5C 5536 NM_ GGGCGUGAGCUGUCAGUUU PPP5C 5536 NM_ GACCAACCCCUAUAUAUUU PPP5C 5536 NM_ UGUACGAGCUCUUUAGCGA PPP5C 5536 NM_ GAUCUACGGUUUCGAGGGU PPP4C 5531 NM_ GCACUGAGAUCUUUGACUA PPP4C 5531 NM_ GACAAUCGACCGAAAGCAA PPP4C 5531 NM_ GCACUUAAGGUUCGCUAUC PPP4C 5531 NM_ GGAGCCGGCUACCUAUUUG Reference Zeng, K., R.N. Bastos, F.A. Barr, and U. Gruneberg Protein phosphatase 6 regulates mitotic spindle formation by controlling the T-loop phosphorylation state of Aurora A bound to its activator TPX2. J. Cell Biol. 191: S5