BIK

BIM

NOXA

PUMA

MCL-1

p53

HCT116 wt cells

DM

SO

Pro

teas

ome

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bito

r I

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xom

icin

ALL

N

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2 E

3 Li

gase

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r

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S

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262

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�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

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IM

�N

OXA

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IK

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IK

�B

IM

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OXA

�PU

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1299

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DMSO

ProteasomeInhibitor I

Epoxomicin

ALLN

Hdm2 E3 LigaseInhibitor

AdaAhX3L3VS

MG262

β-Lactone

α-MOL

DMSO

ProteasomeInhibitor I

Epoxomicin

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Hdm2 E3 LigaseInhibitor

AdaAhX3L3VS

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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

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ivity

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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

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30

20

wt BAX,BAK DKO

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pase

s 3/

7 ac

tivity

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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

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24 36Time, hr

� AIF

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6% 30% 27%

8% 23% 11% 21% 21%

DMSO MG132 Etoposide MG132+zVAD MG132+CsA

wt

BA

X,B

AK

DK

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