Page 42 - Read Online

Bijnsdorp et al. Cancer Drug Resist 2021;4:719-27 https://dx.doi.org/10.20517/cdr.2021.21 Page 721

METHODS
Cell culture and chemicals
The human colon carcinoma cell line Colo320 was obtained from the American Type Culture Collection
and Colo320TP1 was transfected with TP, as described previously . Cells were cultured as monolayers in
[30]
Dulbecco’s Modified Essential Medium supplemented with 10% heat inactivated fetal calf serum and 20 mM
Hepes in 25 cm culture flasks (Greiner Bio-One, Frickenhausen, Germany). Cells were maintained in a
2
humidified 5% CO atmosphere at 37 °C. TPI was provided by Taiho Pharmaceuticals Co. Ltd. (Tokushima,
2
Japan). 3-Methyladenine (3-MA) and TdR were obtained from Sigma Aldrich Chemicals (Zwijndrecht, The
Netherlands). TdR was dissolved in phosphate buffered saline (PBS) in stock solutions of 20 mM.

Drug cytotoxicity assays
[31]
Drug cytotoxicity was determined by the sulforhodamine B (SRB)-assay . Two thousand cells/well were
seeded in 96-well plates (Greiner Bio-One). After 24 h, enabling attachment, cells were exposed to
increasing concentrations of rapamycin for 72 h, with and without 100 µM TdR and/or 10 µM TPI. For
[17]
experiments where 3-MA was used, cells were exposed to 10 mM 3-MA simultaneously with all tested
combinations. After 72 h of drug exposure, cells were precipitated with trichloroacetic acid for 1 h at 4 °C,
colored with SRB and analyzed as described previously . To calculate the growth inhibition curves, optical
[31]
density values were corrected for readings on the day of drug addition. The 50% growth inhibitory
concentration (IC ) values were subsequently determined from graphs and are given as means ± SEM.
50

Fluorescence-activated cell sorting analysis of cell cycle distribution
Cell cycle analysis and apoptosis measurements were performed as described previously . In brief, 200,000
[32]
cells were seeded in six-well plates. After 72 h treatment, cells were trypsinized, resuspended in medium that
was collected from the matching sample and centrifuged for 5 min at 1200 rpm. Subsequently, cells were
stained with propidium iodide (PI) buffer (0.1 mg/mL with 0.1 % RNAse A) in dark on ice. DNA content of
the cells was analyzed by Fluorescence-activated cell sorting (FACS) (Becton Dickinson) with an acquisition
of 10,000 events. The sub-G1 peak was used to determine the extent of cell death.

Western blotting
2
6
Colo320 cells were seeded at 1.5 × 10 in 25 cm culture flasks (Greiner Bio-One). After 6 h, cells were
exposed to 100 µM TdR, 10 µM TPI, or 20 nM rapamycin. After treatment, cells were scraped in lysis buffer
(Cell Signaling Technology, Inc, Danvers, MA, USA) supplemented with 0.04% protease inhibitor cocktail
and centrifuged at 14,000 rpm at 4 °C for 10 min. Protein concentration in the supernatant was determined
by performing a Bio-Rad protein assay according to the manufacturer’s instructions (#500-0006, Bio-Rad
Laboratories, Veenendaal, The Netherlands). From each condition, 30 µg of protein were separated on an
8%-10% SDS-PAGE gel and electroblotted onto a fluorescence polyvinylidene fluoride-membrane
(Millipore Corp., Billerica, MA). Blotted membranes were blocked in Rockland blocking buffer (Rockland,
Tebu-bio, Heerhugowaard, The Netherlands) for 1 h at room temperature (RT) and subsequently incubated
overnight at 4 °C with the primary antibodies directed against Akt (#9272), phospho-Akt, (Ser473 #9271),
p70/S6k (#2708), phospho-p70/S6k (#9205), mTOR (#2983), and phospho-mTOR (#2971) (1:1000; Cell
Signaling Technology) in the Rockland blocking buffer and PBS-Tween solution (0.05% Tween-20).
Antibody targeted against β-Actin (A5441) was used at a 1:10,000 dilution. The membranes were washed
five times in PBS-Tween and incubated with the secondary infrared labeled antibody (1:10,000) for 1 h at
RT in the dark. After incubation, the membrane was washed in PBS-Tween and then 5 min in PBS without
Tween-20 to reduce the background. Subsequently, the bands were scanned using Odyssey Infrared Imager,
with the settings: 84-µm resolution, 0-mm offset, and high quality .
[10]