JCB
T
H
E
J
O
U
R
N
A
L
O
F
C
E
L
L
B
IO
L
O
G
Y
Supplemental material
Espert et al., http://www.jcb.org/cgi/content/full/jcb.201406109/DC1
Figure S1. Bub1 and BubR1 kinetochore localization are regulated by Mps1 and a PP2A-family phosphatase. (A) HeLa cells were spindle checkpoint ar-rested 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.
Figure S2. PP2A-B56 controls BubR1 kinetochore localization and mitotic progression. (A) SAC-arrested HeLa cells depleted of individual PP2A-B56 sub-units 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 kineto-chores per bar). (C) Depletion efficiency of different PP2A-B56 subunits was assessed by Western blotting. (D) Depletion of PP2A-B56 results in mitotic ar-rest. 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.
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
Table S1. Phosphatase SmartPool siRNA sequences
Gene name Gene ID Accession number Target sequence (5 to 3)
PPP2CA 5515 NM_002715 GAACUUGACGAUACUCUAA PPP2CA 5515 NM_002715 GCUUGUAGCUCUUAAGGUU PPP2CA 5515 NM_002715 GGCAAGAUAUUUCUGAGAC PPP2CA 5515 NM_002715 GCAAAUCACCAGAUACAAA PPP2CB 5516 NM_004156 CACGAAAGCCGACAAAUUA PPP2CB 5516 NM_004156 UUUAGUAGAUGGACAGAUA PPP2CB 5516 NM_004156 CCAGAACGCAUUACAAUAU PPP2CB 5516 NM_004156 GAACCAGGCUGCUAUCAUG PPP2R1A 5518 NM_014225 AGGCGGAACUUCGACAGUA PPP2R1A 5518 NM_014225 AAACUUAACUCCUUGUGCA PPP2R1A 5518 NM_014225 GUUCACAGAGCUCCAGAAA PPP2R1A 5518 NM_014225 GAGCUUCUGCCUUUCCUUA PPP2R2A 5520 NM_002717 CAUACCAGGUGCAUGAAUA PPP2R2A 5520 NM_002717 GUAUAGAGAUCCUACUACA PPP2R2A 5520 NM_002717 GCAAGUGGCAAGCGAAAGA PPP2R2A 5520 NM_002717 AGACAUAACCCUAGAAGCA PPP2R2B 5521 NM_181676 UCGAUUACCUGAAGAGUUU PPP2R2B 5521 NM_181676 GGGUCGGGUUGUAAUAUUU PPP2R2B 5521 NM_181676 GAAUGCAGCUUACUUUCUU PPP2R2B 5521 NM_181676 CCACACGGGAGAAUUACUA PPP2R2C 5522 NM_181676 CGGAGGAUCUUUGCCAAUG PPP2R2C 5522 NM_181676 GAUACAACCUGAAGGAUGA PPP2R2C 5522 NM_181676 CCAACAACCUGUACAUCUU PPP2R2C 5522 NM_181676 GAAGAUUACCGAACGAGAU PPP2R2D 55844 NM_001003656 GUAGGUCCUUCUUCUCAGA PPP2R2D 55844 NM_001003656 UCGGAUAGCGCCAUCAUGA PPP2R2D 55844 NM_001003656 GAGACUACCUGUCGGUGAA PPP2R2D 55844 NM_001003656 GAGAACGACUGCAUCUUUG PPP2R5A 5525 NM_006243 GCUCAAAGAUGCCACUUCA PPP2R5A 5525 NM_006243 CAAUACAAGUGCCGAAUAA PPP2R5A 5525 NM_006243 UGAAUGAACUGGUUGAGUA PPP2R5A 5525 NM_006243 GGAAAUGAAUGGCAAGCUU PPP2R5B 5526 NM_006244 CGCAUGAUCUCAGUGAAUA PPP2R5B 5526 NM_006244 UCAAGUCGCUGUCUGUCUU PPP2R5B 5526 NM_006244 CAAACCAUCGUAUCACUGA PPP2R5B 5526 NM_006244 GAACAAUGAGUAUAUCCUA PPP2R5C 5527 NM_178588 GGAUUUGCCUUACCACUAA PPP2R5C 5527 NM_178588 GGAAGAUGAACCAACGUUA PPP2R5C 5527 NM_178588 CAUCAGAAUUUGUGAAGAU PPP2R5C 5527 NM_178588 CAGAAGUAGUCCAUAUGUU PPP2R5D 5528 NM_180977 GUACAUCGACCAGAAGUUU PPP2R5D 5528 NM_180977 UCCAUGGACUGAUCUAUAA PPP2R5D 5528 NM_180977 UGACUGAGCCGGUAAUUGU PPP2R5D 5528 NM_180977 GUAGGCAGAUCAACCACAU PPP2R5E 5529 NM_006246 UUAAUGAACUGGUGGACUA PPP2R5E 5529 NM_006246 GCACAGCUGGCAUAUUGUA PPP2R5E 5529 NM_006246 GACACGCUAUCUGAUCUUA PPP2R5E 5529 NM_006246 GGAUAAAGUAGACGGAUUU PPP1CA 5499 NM_206873 GCAAGAGACGCUACAACAU PPP1CA 5499 NM_206873 GAGCAGAUUCGGCGGAUCA PPP1CA 5499 NM_206873 CAUCUAUGGUUUCUACGAU PPP1CA 5499 NM_206873 GAACGACCGUGGCGUCUCU PPP1CB 5500 NM_206877 GCAAAGUUAUGGUCGAUUU PPP1CB 5500 NM_206877 GCUGAGCGAUUUCUAAUAA PPP1CB 5500 NM_206877 GUGCUUAACUGUCUAAUAU PPP1CB 5500 NM_206877 GAGAAGAGACUUAAUCCAA PPP1CC 5501 NM_002710 GCGGAGAGUUUGACAAUGC PPP1CC 5501 NM_002710 CGAAUUAUGCGACCAACUG
Table S1. Phosphatase SmartPool siRNA sequences (Continued)
Gene name Gene ID Accession number Target sequence (5 to 3)
PPP1CC 5501 NM_002710 UAGAUAAACUCAACAUCGA PPP1CC 5501 NM_002710 UGACAUCCAUGGACAAUAC PPP6C 5537 NM_002721 CUAAAUGGCCUGAUCGUAU PPP6C 5537 NM_002721 CGCUAGACCUGGACAAGUA PPP6C 5537 NM_002721 GUUUGGAGACCUUCACUUA PPP6C 5537 NM_002721 CGAACGGAAUCAGGAAAUU PPP5C 5536 NM_006247 GGGCGUGAGCUGUCAGUUU PPP5C 5536 NM_006247 GACCAACCCCUAUAUAUUU PPP5C 5536 NM_006247 UGUACGAGCUCUUUAGCGA PPP5C 5536 NM_006247 GAUCUACGGUUUCGAGGGU PPP4C 5531 NM_002720 GCACUGAGAUCUUUGACUA PPP4C 5531 NM_002720 GACAAUCGACCGAAAGCAA PPP4C 5531 NM_002720 GCACUUAAGGUUCGCUAUC PPP4C 5531 NM_002720 GGAGCCGGCUACCUAUUUG
Reference
Zeng, K., R.N. Bastos, F.A. Barr, and U. Gruneberg. 2010. 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. 191:1315–1332. http://dx.doi.org/10.1083/jcb.201008106