Chromosome segregation errors: a double-edged sword - TI Pharma

Bioavailability of compound-5 after oral (p.o.) and intravenous (i.v.) application was determined in
Balb/c mice. To determine oral bioavailability a single dose of 10 µmol per kg of compound-5 was
applied as a suspension in 0.5 % gelatin, 5 % mannitol in H 2 O. For the i.v. study the compound was
dissolved in 5% DMSO, 5% cremophor, 5% mannitol in H 2 O and administered at 4 µmol per kg.
For oral administration of 50mg/kg (80 mmol/kg) compound-5, the compound was dissolved at a
concentration of 10mg/ml in the vehicle: 10% DMSO 5% Mannitol and 10% Cremophor.H 2 O. 5ml/
gram mouse was administered orally once a day for 4 consecutive days. On day 4 or 5 mice were
euthanized and tissues were isolated and fixed in formaldehyde for 48 hours. Tissues were rehydrated,
cut into 4 μm sections and stained with Haematoxylin and eosin. For immunohistochemical staining
of ki67, antigen retrieval was performed with citra solution (Biogenex HK086-5K). Before slides
were incubated with ki67 primary antibodies, slides were pre-incubated with 1% milk/PBS. Next,
slides were incubated with HRP-conjugated Envision (Dako) or stained with biotin-conjugated
secondary antibodies and incubated with HRP conjugated streptavidin-biotin complex (Dako).
Following detection with 3,3-diaminobenzidine-tetrahydrochloride (DAB; Sigma A-6926), slides were
counterstained with Haematoxylin and dehydrated. Pictures were taken using an Aperio ImageScope
(Aperio) equipped with a 20x objective and analysis was done using ImageScope software.
For immunofluorescence staining of tissues, antigen retrieval was performed by boiling slides in
Sodium Citrate dehydrate buffer for 20 minutes. Sections were pre-incubated with TBS-0,1% Tween20
4% BSA for 30 minutes at room temperature. Anti-phospho-Histone H3 (1:250) was incubated for 3
hours in TBS-0,1% Tween20 4%BSA, washed twice with TBS-0,1% Tween20 and secondary antibody
was incubated for 45 minutes in the presence of DAPI. Prolong Antifade was used to mount the
slides and analysis was done on a DeltaVision RT system (Applied Precision) with 60x or 10x objective
(Olympus) using SoftWorx software.
Antibodies
The following antibodies have been used for Western Blot and Immunofluorescence: Mouse anti-
Mps1 was from Millipore (05-682). Rabbit anti-Securin was from Abcam (Ab26273), Mouse anti Cyclin
B from Santa Cruz and anti-rabbit Alexafluor568, Rabbit anti-Goat-PO, Goat anti-Mouse-PO and Goat
anti-Rabbit-PO were all from DAKO. Rabbit anti-Phospho Serine10 Histone H3 and anti-MPM2 were
from Upstate.
Acknowledgements
We thank Ellen Mattaar for performing Biacore experiments and data fitting. We thank Antoon van
Doornmalen and Judith Haarhuis for performing cellular assays. We thank Livio Kleij from the University
Medical Center Utrecht for assistance with the microscopes, the personnel of the animal facilities
of the Netherlands Cancer Institute (NKI) for excellent animal husbandry, the NKI animal pathology
department for their expertise on immunohistochemical stainings and Anita Pfauth from the NKI
FACS facility for sorting cells. We also want to thank all the members of the Medema, Lens, Rowland
and Kops labs for helpful discussions. This work was supported by the Netherlands Organisation for
Scientific Research (NWO) (R.H.M. and A.J.: ZonMw 918.46.616; G.J.P.L.K.: VIDI-91776336), TI Pharma
(G.J.R.Z, H.A., B.H., R.H.M and A.J.: T3-105) and the ERC (G.J.P.L.K.: ERC-StG KINSIGN). R.H.M. was
additionally funded by the Netherlands Genomic Initiative of NWO.
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Mps1 inhibition kills aneuploid cells
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