Antibodies and supplementary methods
Cells lines and reagents
SW480 and SW620 human colon carcinoma cell lines were obtained from the
American Type Culture Collection (ATCC). Two derivative SW480 clones
overexpressing human Bcl-2 protein (designated SW480/Bcl-2) and two SW620
clones overexpressing the fusion protein human truncated Bid-GFP (Green
Fluorescent Protein) were generated in the laboratory. They were routinely
maintained in Dulbecco’s minimal essential medium (DMEM) (Gibco)
supplemented with 10% heat-inactivated fetal bovine serum, 1% MEM sodium
pyruvate, 0.1 mg/ml streptomycin, 100 U/ml penicillin at 37°C in a 5% CO 2
humidified atmosphere.
Flag-tagged recombinant soluble human TRAIL from Alexis Biochemicals
(Coger, Paris, France) was used at 25 ng/ml and the anti-Flag (M2) (Sigma
Aldrich; St. Quentin Fallavier, France) at 2µg/ml. ABT-737 was prepared as
described (Oltersdorf et al., 2005) and used at 500nM or 1µM with a 2h preincubation time. Bortezomib was generously donated by Jean-Luc Villeval
(Villejuif, France) and was pre-incubated 2h at 30 nM. Staurosporine was used
at 10µM (Sigma Aldrich).
The following antibodies were used: anti-Bax (N-20, Santa Cruz Biotechnology,
Inc), anti-Bid (R&D SYSTEMES), anti-caspase-3 (H-277, Santa Cruz
Biotechnology, Inc), anti-caspase-8 (Alexis), anti-caspase-9 (Cell Signaling),
anti-Hsc70 (B-6, Santa Cruz Biotechnology, Inc), anti-cytochrome oxidase
subunit IV (anti-COX IV) (Abcam), anti-FADD (BD Transduction
Laboratories), anti-Mcl-1 (S-19, Santa Cruz Biotechnology, Inc), anti-Bcl-2
(4D7, Merck Biosciences Ltd), anti-Bcl-xL (BD Transduction Laboratories),
anti-β-tubulin (clone TUB 2.1, Sigma Aldrich), anti-DR5 (Cayman chemical),
anti-Smac/Diablo (Abcam), anti-cytochrome c (C-20, Santa Cruz
Biotechnology, Inc), anti-Bak (Ab-2, Merck Biosciences Ltd), anti-survivin
(Novus biologicals), anti-XIAP (BD Transduction Laboratories); for flow
cytometry, anti-DR4 and anti-DR5 (Alexis Biochemicals), anti-DcR1 (B-D44)
and anti-DcR2 (B-R27) (provided by Diaclone), anti-Bax (clone 6A7, Sigma
Aldrich) and anti-Bak (Ab-2).
Flow cytometry analysis
Apoptosis was detected by staining permeabilized cells with propidium iodide as
already described (Paris et al., 2007).
Cell surface expression of DR4, DR5, DcR1 and DcR2 was detected after
incubation of cells with 10µg/ml of primary antibody before washing and
incubating with 488-Alexa (green) goat anti-mouse IgG (H+L) secondary
antibody (Molecular Probes).
For Bax and Bak activation measurement, washed cells permeabilized in 0,05%
saponin were incubated with monoclonal anti-active Bax (clone 6A7, SigmaAldrich) raised against a peptide corresponding to amino acids 1-21 of human
Bax; or in mouse monoclonal anti-active Bak (Ab-2). The cells were rinsed and
reincubated with Alexa Fluor 488 goat anti-mouse IgG (H+L) conjugate.
Western blotting
For XIAP, Bcl-2, Bcl-xL, Mcl-1, survivin, Bax and Bak detection cells were
lysed in NP-40 lysis buffer (30 mM Tris pH 7.5, 1% NP-40, 10% glycerol, 150
mM NaCl, 10 mM NaF, 1 mM sodium orthovanadate, 1 mM
phenylmethylsulfonylfluoride (PMSF), 10 µg/ml aprotinin and 10 µg/ml
leupeptin). For detection of caspases, cells were lysed in Laemmli buffer (60
mM Tris pH 6.8, 10% glycerol and 2% SDS) and lysates were sonicated gently
on ice.
All lysates were clarified by centrifugation at 15000 rpm for 15 min at 4°C.
Protein concentration was assessed using the Bicinchoninic Acid (BCA) assay
kit (Sigma Aldrich). 50 g of protein were incubated in loading buffer (60 mM
Tris-HCl, pH 6.8, 0.18M β-mercaptoethanol, 2% SDS, 10% glycerol, and
0.005% bromophenol blue), boiled and separated on a polyacrylamide gel by
SDS-PAGE. After overnight transfer to polyvinylidene difluoride (PVDF)
membrane (Hybond-P, Amersham Biosciences) and blocking for at least 1h in
Tris-buffered saline (TBS), 5% BSA and 0.5% Tween 20, membranes were
probed with the appropriate antibodies and proteins were visualized by enhanced
chemiluminescence (ECL, Amersham Pharmacia Biotech.).
Immunofluorescence
Bax relocalization and activation analysis: After 24h of stimulation with TRAIL,
cells were harvested, washed twice with complete medium and the cell
suspension was added on glass coverlips coated with 0,002% poly-L-lysine
(Sigma-Aldrich) for 1h. Cells were permeabilized with 0.05% saponin and
incubated with the primary antibody mouse monoclonal anti-Bax (clone 6A7).
The coverlips were washed and cells were then incubated with 546-Alexa (red)
goat anti-mouse IgG. After washing coverlips were mounted onto microscope
slides using Mowiol. Cells were subsequently viewed by confocal microscopy.
To ascertain localization of the mitochondria, cells were stained with
mitoTracker Red CMXRos dye (MitoTracker Red, Molecular Probes,
Invitrogen). A merged double image of green GFP and MitoTracker Red was
obtained using a confocal laser scanning microscope, with excitation at 488 and
568 nm, and with emissions filtered using a double-window barrier filter, of
which the ranges were 510-590 nm and 580-620 nm.