Supplementary information
MATERIAL AND METHODS
Western blotting
Cells were lysed in 500 µl of lysis buffer (50 mM Tris/HCl, pH 7.4, 1% (v/v) Triton X-100,
150 mM NaCl, 1 mM EDTA, 0.1 mM PMSF and 0.1 mM Na3VO4) supplemented with
protease inhibitor cocktail (CompleteTM-Mini/EDTA free; Roche) and further treated three
times with sonication for 10 s. For preparation of nuclear extracts, tissue homogenates or cells
were lysed and prepared as described [Arlt et al. 2003]. After adjusting to equal protein
concentrations (using the Dc-Protein assay; Bio-Rad, München, Germany), the samples were
supplemented with the half volume of 3×SDS/PAGE sample buffer, heated (95°C) for 5 min
and submitted to 8–20% PAA (polyacrylamide agarose)-gradient electrophoresis. Upon semidry transfer on to a PVDF membrane, blots were blocked for 2h with 5% non-fat milk powder
and 0.05% Tween20 in PBS (blocking solution) at room temperature. Afterwards, blots were
exposed to the primary antibodies against S5a/PSMD4/Rpn10 and alpha5/PSMA5 (both from
Affiniti/Biomol, Hamburg, Germany), against cIAP1 and cIAP2 (both R&D Systems), against
caspase-3 (Cell Signaling), against HA-tag (Sigma) and against Mcl1, Nrf2 and Hsp90 (all
from Santa Cruz Biotechnology, Heidelberg, Germany) at a 200- to 1000-fold dilution in
primary antibody diluent (5% (w/v) non-fat milk powder, 0.05% Tween20 in TBS (Trisbuffered saline; 50 mM Tris/HCl, pH 7.6, and 150 mM NaCl)) overnight at 4°C. After
extensive washing with 0.05% Tween20 in TBS, blots were exposed to the appropriate horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology) diluted
(1:1000) in blocking buffer and were developed using the Dura detection kit (Perbio Sciences,
Bonn, Germany).
Fluorometric proteasome assay. Cells were harvested by mild trypsinization. Then, 5×105
cells were resuspended in 500 µl medium and incubated with 5 µ/mL of the proteasome
inhibitor MG-132 (Biomol, Hamburg, Germany) or not. After 30 min, 5 µl of Suc-LLVYAMC (from Biomol, 5 mM dissolved in DMSO) was added, and at various time points the
formation of the fluorescent dye AMC was measured at =460 nm using a
spectralfluorometer (Infinite F200, Tecan). Specific chymotryptic proteasomal activity was
determined by subtraction of the mostly proteasome-independent fluorescence, measured in
the presence of MG-132. These OD-values were normalized to cell numbers determined in
parallel by flow cytometry.
For the detection of proteasome activity in frozen tissue, ground tissue (see above) was
resuspended in lysis buffer (50 mM potassium phosphate pH 7.4, 5 mM MgCl2, 0.1% TritonX100, 5% glycerol) and subjected to three freeze and thaw cycles. Cell debris was removed
by centrifugation at 10,000 rpm, 4°C for 5 min and the supernatant was adjusted to equal
protein amounts (Dc-Protein assay; Bio-Rad, Munich, Germany). One hundered µl of these
extracts were mixed with 400 µl AMC assay buffer (50 mM potassium phosphate pH 7.4, 2
mM MgCl2, 1.25 mM DTT, 5% glycerol, 10 mM ATP supplemented with 5 µl Suc-LLVYAMC) and AMC dye formation in the presence or absence of 5 µg/mL MG132 was measured
as described above.
Real-time PCR. The primer sequences and the PCR conditions for the detection of the
proteasomal subunits were: 5´-GAAGGTGGCAAGATGGTGTTGGA-3´ (S5a/psmd4, sense)
and 5´-AGGCACTGTCACCAGATGAGAAC-3´ (S5a/psmd4, antisense); 5´-GCCATTG
AGGCTATCAAGCT TGC-3´ (alpha5/ psma5, sense) and 5´-AACTTCTTGCAAGGAGCTC
TGGG-3´ (alpha5/psma5, antisense); 95°C for 2 min; 40 cycles 95°C for 1 min, 60°C for 30
sec, 72°C for 30 sec. The primer sequences and the PCR conditions for the detection of antiapoptotic NF-B target genes were: 5´-GCCTGATGCTGGATAACTGG-3’ (cIAP1, sense)
and 5’GGCGACAGAAAAGTCAATGG-3’ (cIAP1, antisense); 5´-GCCTGATGCTGGAT
AACTGG-3’ (cIAP2, sense) and 5’GCTCTTGCCAATTCTGATGG-3’ (cIAP2, antisense);
95°C for 2 min; 40 cycles 95°C for 1 min, 55°C for 30 sec., 72°C for 30 sec. Mcl-1 was
detected using primers from R&D-Sytems following the manufacturer’s protocol and -actin
was amplified for normalization of mRNA content using primers from Lonza (Verviers,
Belgium). The real-time PCR was performed with a MyiQ Single Color Real-time PCR
Detection System (Bio-Rad). Data were collected during annealing steps and were further
analysed by using the iCycler iQ optical system software (Bio-Rad). All samples were
analysed in duplicate and data are expressed as amount of mRNA normalized to actin. The
quality of PCR products was checked by melting curve analysis following the amplification as
well as by analytical RT (reverse transcriptase)–PCR and subsequent gel electrophoresis.
Supplementary Fig. 1
Legend to supplementary figure 1
Densitometrically determined quantities of S5a and nuclear Nrf2 protein
levels (normalized to -actin and Hsp90, respectively) were correlated to the
specific AMC formation/mg protein from matched normal and tumoral
tissue, or to each other.
T
N
N
T
N
T
N
T
T
N
N
T
T
N
T
N
T
N
T
N
N
T
T
N
N
T
T
N
N
T
T
N
N T
T
NFB
P3
P4
P3
P6
P4
P7
P6
P7
P10
P10
P12
P19
P12
P19
P20
P20
P21
P21
Supplementary Fig.
2 1E
Supplementary
Fig.
Supplementary Fig. 1E
LegendLegend
to supplementary
figure 21E
to supplementary figure 1E
NuclearNuclear
extracts
of
normal
and
tumoral
from9 9(out
(out
of 21)
extracts of normal and
tumoraltissues
tissues from
of 21)
coloncolon
cancercancer
patientspatients
(numbering
refers
to those
depicted
in Figure
(numbering
refers
to thosecase
casenumbers
numbers depicted
in Figure
1a-c)1a-c)
were were
analysed
by
NF-B
gel
shift
asssay.
From
the
other
patients
no
sufficient
amounts
analysed by NF-B gel shift asssay. From the other patients no sufficient amounts
of nuclear extracts of the matched tissues were available.
of nuclear
extracts of the matched tissues were available.
NCM460 + tBHQ
NCM460 + tBHQ
9
8
7
6
5
4
3
2
vehicle
TRAIL
MG 132
TRAIL+MG132
80
AnnexinV positive cells (%)
10
Casapse-3/-7 activity (n-fold)
N
vehicle
TRAIL
MG 132
TRAIL+MG132
70
60
50
40
30
20
1
10
0
0
Supplementary Fig. 3
Legend to supplementary figure 3
tBHQ treated NCM460 cells were incubated for 1h with 5 µM MG132 or without followed
by administration of 0.1 µg/mL TRAIL for 16 h, or not. Apoptosis was determined by
Casapse-3/7 assay and AnnexinV/PI assay. Data represent the mean of two independent
experiments.