BIK BIM NOXA PUMA MCL-1 p53 HCT116 wt cells DMSO Proteasome Inhibitor I Epoxomicin ALLN Hdm2 E3 Ligase Inhibitor AdaAhX3L3VS DMSO MG262 β-Lactone α-MOL DMSO Proteasome Inhibitor I Epoxomicin ALLN Hdm2 E3 Ligase Inhibitor AdaAhX3L3VS MG262 β-Lactone α-MOL Procaspase 3 PARP Cleaved Product 48 48 48 48 48 48 48 48 48 24 24 24 24 24 24 24 24 24 hr 48 hr 0 40 80 120 Viability, % MG132 PSI Epoxo ALLN Hdm2 LI MG262 lactone Ada 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.
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BIK
BIM
NOXA
PUMA
MCL-1
p53
HCT116 wt cells
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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
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Viab
ility
, %
MG132
PSIEpo
xoALL
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Hdm2 L
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MG262
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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
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SOMG132 MG132 MG132 MG132
48 48 4848 48 48 484824 24 24 24 hr
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MG
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MG
132
MG
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MG
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48 hr
Saos2 H1299 C-33A LoVo0
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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.
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ProteasomeInhibitor I
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DMSO
ProteasomeInhibitor I
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AdaAhX3L3VS
MG262
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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
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
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30C
ell D
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eter
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e U
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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
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anti-Bak #5
MG132, mM0.1 0.5 1 2 5
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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
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V-P
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C
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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.