6730
S. Pédeboscq et al. / Bioorg. Med. Chem. 20 (2012) 6724–6731
4.1.5.3.2. N-(2-methylbenzyl)-benzo[4,5]thieno[2,3-d]pyrimidin-4-
amine (7b). Compound 7b (R = CH3): mp: 164–166 °C; yield 70%;
IR KBr, cmꢀ1: 3417( NH); 1H NMR (300 MHz, DMSO-d6): d ppm
2.39 (s, CH3), 4.83 (s, CH2), 7.14 (m, 5H), 7.57 (t, J = 8.4 Hz, 1H,
H6), 7.99 (s, NH), 8.09 (d, J = 7.2 Hz, 1H, H8), 8.44 (s, 1H, H2),
8.63 (d, J = 7.2 Hz, 1H, H5; 13C NMR (75 MHz, DMSO-d6): d ppm
19.3 (CH3), 34.4 (CH2), 110.5 (C4a), 123.6 to 126.8 (C5 to C8 and
C14 to C16), 130.2 (C4b), 131.8 (C13), 134.6 (C12), 135.5 (C8a),
137.7 (C11), 155.5 (C2), 157.3 (C4), 167.7 (C8b).
temperature. After 3 washes in PBS, cells were permeabilized in
0.5% Triton X-100 for 5 min. Nonspecific binding was blocked by
incubating cells in 0.2% gelatin/PBS for 30 min at RT, and staining
was performed using a specific primary antibody anti-EGFR (clone
31G7, Zymed laboratories) with a dilution 1/100 overnight at 4 °C
and a fluorescent secondary antibody conjugate (Alexa Fluor 488
(green), Molecular Probes) for 1 h at RT. Nuclear staining was sub-
sequently done with a 1/5000 dilution of Hoechst 33258 (10 mg/
mL) (Molecular Probes) for 10 min at RT. Coverslips were washed
once in PBS and then mounted with Fluoromount G on glass slides.
Image acquisition was performed using a Zeiss LSM 510 Meta
microscope.
m
4.2. Biological assays
4.2.1. Cell culture
Colorectal carcinoma cell lines HT29 and HCT116, and Hela cell
line were cultured in RPMI 1640 containing 10% heat-inactivated
FCS supplemented with sodium pyruvate (1 mM) and glutamax
1X. Cells were cultured in 75 cm2 flasks and maintained at 37 °C
in a fully humidified 5% CO2 atmosphere. Subcultures were as-
sessed by using trypsin/EDTA solution (1:250) for 5 min at 37 °C.
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
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mode (515–545 nm) for GFP and in FL3 log mode (>650 nm) for
TMRM. Forward scatter (FSC) and side scatter (SSC) were used to
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fluorescent and non-fluorescent cells on each mode. The number
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4.2.5. Immunofluorescence
Cells were seeded onto glass coverslips in 96-well plates and
fixed 24 h later with 3.7% formaldehyde for 10 min at room