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

Supplemental Figure-1.
Protein biochemistry
For the Biacore experiments all reagents were obtained from GE Healthcare unless stated otherwise.
Direct binding experiments were carried out using a Biacore T100 instrument. Full length Mps1
(Invitrogen Cat. No: PV3792) was immobilized on a CM5 sensor chip using common amine coupling:
The surface is activated for 900 seconds using a 1:1 dilution of 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)
carbodimide hydrochloride (EDC) and 50 mM N-hydroxysuccinimide. Full length Mps1 was injected
for 1800 seconds diluted to 45 µg/ml in 10 mM Tris, 10 mM MgCl 2 , 0.01% Tween-20 and 1 mM DTT
at pH 7.2. Remaining active groups on the surface were blocked by a 420 second injection of 1M
ethanolamine. A reference surface was generated using a similar procedure without the injection of
Mps1. These injections usually result in the immobilization of approximately 10.000 RU (Response
Units). Direct binding method: Low molecular weight compounds were diluted in the described buffer
resulting in an end concentration of 1% DMSO. An injection series of 5 increasing concentrations
(usually: 0.3, 0.9, 2.7, 8.3 and 25 nM –or a series of 10 times higher concentrations) were injected on
a freshly immobilized surface as well as a control surface, at a flow rate of 50 µl/min. This method is
known as single cycle kinetics. The dissociation after the last injection was monitored for at least 1800
seconds. No regeneration was carried out and each compound was tested on a freshly immobilized
surface. Using Biacore T100 evaluation software v. 2.0.1 the measured data were fitted to a simple 1:1
Langmuir binding model, after subtraction of the average of at least two preceding blanks on the blank
flow cell subtracted data, a method that is known as double reference subtraction; (FCactive –FCblank)
sample - (FCactive –FCblank)blank. The results are evaluated by assessment of the residual plots, the
chisquare and U (uniqueness) value of the fit as well as the sensibility of the calculated kinetic values
in combination with the tested concentrations.
Western Blot analysis
Cells were lysed in Laemmli buffer. Samples were separated by SDS-page and transferred to PVDF
(Immobilin FL, Millipore). The membranes were cut in half and blotted with anti-Mps1, Cyclin B1 and
Securin. Peroxidase-coupled secondary antibodies and ECL (GE healthcare) were used to visualize
protein bands.
Cell culture, cell lines & reagents
All tumor cell lines were grown in DMEM (Lonza), BJ-Tert and RPE-1 cells were grown in DMEM/F-12
+ Glutamax (GIBCO) supplemented with 6% FCS (Clontech), pen/strep (Invitrogen) and ultraglutamine
(Lonza). Taxol, nocodazole, thymidine, MG132 and Staurosporine were all from Sigma. SW480, MCF-7
and RPE-1 cells 389 were infected with retrovirus carrying pBabeH2B-RFP (kind gift from Dr. Susanne
Lens, University Medical Center Utrecht, The Netherlands) and/or pLNCX2GFP-m53BP1 (kind gift from
Dr.Marcel van Vugt, University Medical Center Groningen, The Netherlands). Cell lines were selected
with 5mg/ml blasticidine (for H2B-RFP) and 500mg/ml G418 (for GFP-53BP1). Single colonies were
selected after replating 1-2 cells/well or using FACS sorting. MCF-7 cells stably expressing 53BP1-GFP
were a kind gift from Dr. Marcel van Vugt. BJ-Tert WT, p53KD and p53KD + HRASV12 were all kindly
provided by Dr. Roderick Beijersbergen (Netherlands Cancer Institute, Amsterdam, The Netherlands).
BJ-Tert stably expressing p53KD and HRASV12 were transduced with retrovirus carrying pBabe-E1A
(kind gift from Dr. Benjamin Rowland, Netherlands Cancer Institute, The Netherlands) and selected
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Mps1 inhibition kills aneuploid cells
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