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merization in a high-molarity PIPES buffer (1mK-PIPES, pH 6.9,
2 mm EGTA and 1 mm MgCl2), and protein concentration was de-
termined by the MicroBCA assay kit (Pierce). The test the effects of
the thiocolchicine derivatives, the compounds were dissolved in di-
methyl sulfoxide (DMSO), added in different concentrations (1–
10 mm) to a reaction mixtures (20 mm tubulin, 10% glycerol, 1 mm
GTP in BRB80 buffer) and incubated for 30 minutes at 378C. As
control conditions were used either unmodified thiocolchicine or
DMSO alone. At the end of polymerization, unpolymerized and
polymerized fractions of tubulin were separated by centrifugation
at 16500xg for 30 minutes at 258C. The collected microtubules
were resuspended in SDS-PAGE sample buffer (2% w/v SDS,
10% v/v glycerol, 5% v/v b-mercaptoethanol, 0.001% w/v bromo-
phenol blue, and 62.5 mm Tris, pH 6.8) and the unpolymerized tu-
bulin was diluted 3:1 with 4X SDS-PAGE sample buffer. Equal pro-
portions of each fraction were resolved by a 7.5% SDS-gel and
stained with Coomassie blue. Densitometric analyses of stained
gels were performed by using ImageJ software (National Institute
of Health), and data were elaborated using STATISTICA (StatSoft
Inc., Tulsa, OK). Significant differences were assessed by one-way
ANOVA with Tukey HSD post hoc test. Experiments were done in
triplicate and data are expressed as meansÆSEM.
Keywords: chephalomannines
binders · osmium · thiocolchicines · tubulins
· fluorescence · fluorescent
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Cell culture and fluorescence microscopy
Human lung carcinoma cell line A549 (CCL-185; American Type
Culture Collection, Rockville, MD, USA) was grown in minimal es-
sential medium with Earle’s (E-MEM), supplemented with 10% fetal
bovine serum (Hyclone Europe, Oud-Beijerland, Holland), 2 mm l-
glutamine, 100 UmLÀ1 penicillin, and non-essential amino acids.
Cells were maintained at 378C in a humidified atmosphere at 5%
CO2. To test the ability of thiocolchicine derivatives to be incorpo-
rated into the cells and bind microtubules A549 cells
(50000 cellmLÀ1) were seeded on glass coverslips and grown in
control medium. After 24 h, cells were either incubated 1 hour
with 10 mm thiocolchicine derivatives and then fixed or vice versa,
that is, cells were fixed and then incubated 1 hour with the fluores-
cent compounds. At the end of the treatments, cells were fixed
and permeabilized for 10 minutes with methanol at À208C,
washed with PBS, and blocked in PBS+5% bovine serum albumin
(BSA) for 15 minutes at room temperature. To localize tubulin, the
cells were incubated with monoclonal anti-a-tubulin antibody
(clone B-5–1–2, Sigma–Aldrich) 1:500 in PBS for 1 h at 378C. We
used goat anti-mouse AlexaFluor594 (Molecular Probes) 1:1000 in
PBS+1% BSA for 45 minutes at 378C as secondary antibodies.
Nuclei staining was performed by incubation with DAPI
(0.25 mgmLÀ1 in PBS) for 15 minutes at room temperature. The cov-
erslips were mounted in Mowiol (Calbiochem)-DABCO (Sigma–Al-
drich) and examined with
a Zeiss Axiovert 200 microscope
equipped with a 63x Neofluor lens (Zeiss, Oberkochen, Germany)
and confocal laser scan microscope imaging system (TCS SP2
AOBS, Leica Microsystems, Heidelberg, Germany) equipped with an
Ar/Ar-Kr 488 nm, 561 nm and 405 nm diode lasers.
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Acknowledgements
This research has been developed under the umbrella of CM0602
COST Action “Inhibitors of Angiogenesis: design, synthesis and
biological exploitation”.
Received: September 28, 2012
Revised: November 30, 2012
Published online on January 14, 2013
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemPlusChem 2013, 78, 222 – 226 226