Post on 03-Feb-2022
BIK
BIM
NOXA
PUMA
MCL-1
p53
HCT116 wt cells
DM
SO
Pro
teas
ome
Inhi
bito
r I
Epo
xom
icin
ALL
N
Hdm
2 E
3 Li
gase
Inhi
bito
r
Ada
AhX
3L3V
S
DM
SO
MG
262
β-La
cton
e
α-M
OL
DM
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teas
ome
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r I
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N
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3 Li
gase
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r
Ada
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3L3V
S
MG
262
β-La
cton
e
α-M
OL
�Procaspase 3
�PARP�Cleaved Product
48 48 48 48 48 48 48 48 4824 24 24 24 24 24 24 24
24 hr
48 hr
0
40
80
120
Viab
ility
, %
MG132
PSIEpo
xoALL
N
Hdm2 L
I
MG262
lacton
eAda
MOL
A
B
C Figure S1. HCT116 wt cell death by different proteasome inhibitors.
A.Western blot analysis of BIK, BIM, NOXA, PUMA, MCL-1 and p53 expression at 24 hr after treatment with indicated PIs.
B.Effect of indicated PIs on caspase-3 activation and PARP cleavage.
C.Effect of indicated PIs on cell viability. Viability was determined by the MTT method. The data are expressed as mean±SD from three independent experiments.
�Procaspase 3
�PARP�Cleaved Product
�Activated caspase 3
�BIK
�BIM
�NOXA
�PUMA
�MCL-1
�p53
Saos2 H1299 C-33A LoVo
Saos2 H1299 C-33A LoVo
DM
SO
DM
SO
DM
SO
DM
SOMG132 MG132 MG132 MG132
48 48 4848 48 48 484824 24 24 24 hr
DM
SO
DM
SO
DM
SO
DM
SO
MG
132
MG
132
MG
132
MG
132
24 hr
48 hr
Saos2 H1299 C-33A LoVo0
25
50
75
100
Viab
ility
, %
A
B
C Figure S2. MG132-induced death in different cell lines.
A.Western blot analysis of BIK, BIM, NOXA, PUMA, MCL-1 and p53 expression at 24 hr after treatment with MG132 in Saos2, H1299, C-33A, LoVo cell lines.
B.Effect of MG132 on caspase-3 activation and PARP cleavage in indicated cell lines.
C.Effect of MG132 on viability of indicated cell lines. Viability was determined by the MTT method. The data are expressed as mean ± SD from three independent experiments.
�B
IK
�B
IM
�N
OXA
�PU
MA
�M
CL-1
�p53
�B
IK
�B
IM
�N
OXA
�PU
MA
�M
CL-1
�p53
�B
IK
�B
IM
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OXA
�PU
MA
�M
CL-1
�B
IK
�B
IM
�N
OXA
�PU
MA
�M
CL-1
Saos2H
1299
C-33A
LoVo
DMSO
ProteasomeInhibitor I
Epoxomicin
ALLN
Hdm2 E3 LigaseInhibitor
AdaAhX3L3VS
MG262
β-Lactone
α-MOL
DMSO
ProteasomeInhibitor I
Epoxomicin
ALLN
Hdm2 E3 LigaseInhibitor
AdaAhX3L3VS
MG262
β-Lactone
α-MOL
DMSO
ProteasomeInhibitor I
Epoxomicin
ALLN
Hdm2 E3 LigaseInhibitor
AdaAhX3L3VS
MG262
β-Lactone
α-MOL
DMSO
ProteasomeInhibitor I
Epoxomicin
ALLN
Hdm2 E3 LigaseInhibitor
AdaAhX3L3VS
MG262
β-Lactone
α-MOL
Figure S3. Induction of BH
3-only proteins and p53 by different proteasome inhibitors
in different tumor cell lines.
Western blot analysis of B
IK, B
IM, N
OX
A, P
UM
A, M
CL-1 and p53 expression at 24 hr after treatm
ent w
ith indicated PIs in S
aos2, H1299, C
-33A, LoVo cell lines.
Pull-down: Ub-matrix
WB: MCL-1 p53 NOXA PUMA BIM BIK
**
*
*
* * *
1 - DMSO 2 - MG132
1 1 1 1 1 122 2 2 2 2
A
DMSO MG132Gapdh
Gapdh
Mcl-1lMcl-1l
Mcl-1sMcl-1s
Puma
Puma
Noxa
Noxa
Bim
Bik
Tp53
tv 2
tv 1
tv 7
Bim tv 7
Bim tv 2
Bim tv 1
Bik
Tp53
0 1 2 3 4Fold of Increase
B
DMSO MG132
p53-luc p53-lucpCis-CK pCis-CKHCT116 wt HCT116 p53-/-
Rel
ativ
e Lu
cife
rase
Act
ivity
0
1
2
3C
�E2F-1
�Actin
DM
SO
DM
SO
MG
132
MG
132
p53 p53+/+ -/-D
E2F-1 wt E2F-1 wt E2F-1 mtE2F-1 mt
p53 +/+ p53 -/-
DMSOMG132
Rel
ativ
e Lu
cife
rase
Act
ivity
0
0.01
0.02
0.03
0.04
E
Figure S4. Transcriptional and post-transcriptional regulation of BH3-only proteins and p53.A. Effect of MG132 on the ubiquitination of BH3-only proteins and p53.HCT116 cells were treated with MG132 or DMSO for 16 hr and subjected to a pull-down assay using the ubiquitin affinity matrix. The proteins associated with the matrix were detected by Western blot analysis using antibodies against the indicated proteins.B. Effect of MG132 on BIK, BIM, MCL-1, NOXA, PUMA and p53 mRNA levels.A representative experiment is shown in the left panel. Right panel represents values of mRNA levels induced by MG132 expressed as mean fold increase ±SD relative to vehicle-treated cells; n=3.C. Transcriptional activity of p53 in HCT116 wt and p53-/- cells treated with MG132 or DMSO. The graph represents means of relative transcriptional activity of p53 as determined by the luciferase assay in three independent experiments done in duplicate, bars correspond to SD.D. Western blot analysis of E2F-1 expression in HCT116 wt and p53-/- cells treated with MG132 or DMSO for 24 hr. E. Transcriptional activity of E2F-1 in HCT116 wt and p53-/- cells treated with MG132 or DMSO. The graph represents means of relative transcriptional activity of E2F-1 as determined in C.
DMSO
MG132
HCT116 wtDMSO, 6 hr
HCT116 wtMG132, 6 hr
HCT116 #5MG132, 6 hr
wt #50
10
20
30C
ell D
iam
eter
, Rel
ativ
e U
nits
A
B C
Figure S5. Morphologic features of apoptosis induced by MG132 in HCT116 cell lines.
A.Transmission electron microscopic images of DMSO (left panel) or MG132-treated HCT116 wt (middle panel) or HCT116Bax-/-Bak� #5 (right panel) cells at 6 hr. Note, that exposure to MG132 resulted in a distinctly dilated ER. Bars = 5 μm.
B.High magnification image of MG132-treated cells shows dilated rough ER delimited by electron-dense ribosomes. Bar = 0.5 μm.
C.Analysis of cellular sizes in light microscope images of HCT116 wt and Bax-/-Bak� #5 cell lines at 24 hr after treatment with DMSO or MG132.Values for major and minor axes were acquired for 26 individual cells in each of three separate images to calculate an average diameter of cells. Data are means ± SD.
Awt
Bax-/-
anti-Bak #5
MG132, mM0.1 0.5 1 2 5
Viab
ility
, %
0
50
100
150
200
0.01 0.05 0.1 0.2 0.5Epoxo, μM
Viab
ility
, %
0
50
100
150
200
0
25
50
75
100
0 24 48 72Time, hrsC
ell v
iabi
lity,
%
Bax-/-Bcl-2 #35�BCL-2
�BAK
Bax-/- Bcl-2 #35Bax-/- Bcl-2 #35
DMSO
MG132
B C D
0
25
50
75
100
0 24 48 72
Time, hrs
Viab
ility
, %
MG132MG132+zVAD-FMK
0
25
50
75
100
0 24 48 72
Time, hrs
Viab
ility
, %
MG132MG132+zVAD-FMK
HCT116 Bax-/- Bak� #5HCT116 Bax-/-E
Figure S6. PIs – induced cell death in HCT116 cell lines.
A. HCT116 cell lines were treated with MG132 or Epoxo as indicated and at 48 hr after exposure cell death were determined by MTT assay. Data are expressed as mean ± SD from three independent experiments.B. Western blot analysis of BCL-2 expression in HCT116Bax-/- or HCT116Bax-/-Bcl-2 #35 cells. Level of BAK shown to demonstrate equal protein loading.C. Representative light microscopic (120X) images of HCT116Bax-/- or HCT116Bax-/-Bcl-2 #35 cells at 24 hr after treatment with DMSO or MG132.D. MG132-induced death. Cell viability was determined by MTT assay and expressed as mean percent ± SD relative to vehicle-treated cell lines. Data are from tree independent experiments.E. Effect of zVAD-FMK on MG132-induced death in HCT116 cell lines. HCT116 cell lines were treated with MG132 alone or in combination with 50μM zVAD-FMK. Cell viability was determined at indicated times after exposure by MTT assay and expressed as mean percent ± SD relative to vehicle-treated cell lines. Data are from three independent experiments.
Ann
exin
V-P
ositi
ve, % DMSO
C
DM
SO
A
Figure S7. MG132-induced cell death in BAX/BAK DKO MEFs.A. Viability of wt and BAX,BAK DKO MEFs treated with DMSO or MG132 alone or in combination with zVAD-fmk or CsA at 48 hr after exposure determined by the MTT assay. Results are expressed as mean+/- SD relative to vehicle-treated cells, n=3.B. Caspase 3/7 activity in wt and BAX,BAK DKO MEFs at 24 hr after treatment with DMSO, MG132 and zVAD-fmk or CsA as measured with caspase-Glo 3/7 kit (Promega).C. Analysis of apoptosis in BAX,BAK DKO MEFs. Cells were treated with DMSO, MG132 and zVAD-fmk. Annexin V-positive cells were analysed by flow cytometry. Data are mean values +/- SD from three independent experiments.D. Western blot analysis of cytochrome c and AIF in cytosolic and mitochondrial fractions of BAX,BAK DKO MEFs treated as indicated. Markers for cytosolic and mitochondrial fractions are also indicated.E. Effect on ΔΨm. wt and BAX,BAK DKO MEFs were treated with DMSO, MG132 and zVAD-fmk or CsA and ΔΨm was measured by flow cytometry using Rh123 at 16 hr after treatment.
MG132
B
0
10
30
20
wt BAX,BAK DKO
Cas
pase
s 3/
7 ac
tivity
, RLU
0.E+00
5.E+05
1.E+06
2.E+06
D
DMSO
MG132
MG132+
zVAD
MG132+
CsAzV
ADCsA
wt BAX,BAK DKO
DMSO
MG132
MG132+
zVAD
MG132+
CsAzV
ADCsA
Viab
ility
, %
0
50
100
150
E
MG132+zVAD
24 36Time, hr
� AIF
� Cyt c
� COX IV
� Procaspase 3
DM
SO
MG132 MG132Eto
posi
de
Eto
posi
de
+zV
AD
+zV
AD
+CsA
+CsA- -
Cytosol Mitochondria
6% 30% 27%
8% 23% 11% 21% 21%
DMSO MG132 Etoposide MG132+zVAD MG132+CsA
wt
BA
X,B
AK
DK
O