Supplementary Information
Synergistic effects of nelfinavir and bortezomib
on proteotoxic death of NSCLC and multiple myeloma cells
Shigeru Kawabata, Joell J. Gills, José R. Mercado-Matos,
Jaclyn
LoPiccolo,
Willie
Wilson
III,
M.
Christine
Cancer
Research,
Hollander, and Phillip A. Dennis
Medical
Oncology
Branch,
Center
for
National Cancer Institute, Bethesda, MD 20892
1
Supplemental Figure S1.
apoptosis.
Enhancement of caspase-dependent
RPMI8226 cells were pre-treated or not with 50
µM Z-VAD for 1 h, followed by treatment with either DMSO,
10 µM NFV, 6.25 nM BZ, or the combination for 24 h.
Cells
were harvested and analyzed by DNA fragmentation and cell
death assays.
PARP cleavage was assessed by immunoblotting
to confirm inhibition of caspases by Z-VAD.
Columns, mean
from at least three separate experiments; bars, SD.
*, p
<0.001.
Supplemental
Bcl-2
Figure
expression,
S2.
and
Combining
increased
NFV
DR5
with
and
BZ
DR4
decreases
expression.
H157 cells were treated with either DMSO, 10 µM NFV, 12.5
nM BZ, or the combination for the indicated times.
The
indicated markers were assessed by immunoblotting.
Supplemental Figure S3.
Combining NFV with BZ activates
stress kinases, but not signaling pathways involved in cell
proliferation.
H157 cells were treated with either DMSO,
10 µM NFV, 12.5 nM BZ, or the combination for the indicated
times.
The
indicated
markers
immunoblotting.
2
were
assessed
by
Supplemental
Figure
S4.
Expression
of
ER
stress
and
apoptosis-markers (A) and cytosolic proteotoxicity-markers
(B) in H157 xenograft tumor lysates after the 5-days of
treatment as described in Materials and Methods (5 mice per
group).
The
indicated
markers
were
assessed
by
immunoblotting followed by densitometry analysis for Figure
6C.
C1: H157 cells treated with 0.1% DMSO, C2: H157 cells
treated with the combination of 10 µM NFV and 12.5 nM BZ
for 48 h.
(C) Expression of ER stress-markers in RPMI8226
xenograft tumor lysates after the 5-days of treatment as
described in Materials and Methods (3 mice per group). The
indicated
markers
were
assessed
by
immunoblotting.
Densitometry was performed using NIH Image software, and
levels of each marker were normalized to GAPDH for each
sample; Columns, mean from all 3 mice examined in RPMI8226
xenografts.
bars, SD.
Supplemental
autophagy.
NFV,
12.5
times.
Figure
S5.
Combining
NFV
with
BZ
enhances
H157 cells were treated with either DMSO, 10 µM
nM
BZ,
or
the
combination
for
the
indicated
Levels of LC-3 I and II as a marker of autophagy
were assessed by immunoblotting.
3
Supplemental Figure S6. Concurrent or Sequential schedule
of the treatment with combination of NFV and BZ.
Growth
inhibition by NFV (■), BZ (▲), or the combination (●).
H157
cells
were
treated
with
drugs
at
the
indicated
concentrations for 72 h (A) and 48 h (B), respectively on
the concurrent schedules.
(C) Sequential schedules.
H157
cells were treated with BZ for 24 h followed by additional
NFV for 48 h or with NFV for 24 h followed by additional BZ
for 48 h at the indicated concentrations.
N: NFV; B: BZ;
A: analysis by the sulforhodamine B assay as described in
Materials and Methods.
D1: day 1.
4