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Supplementary Figure Legends
Supplementary Figure S1. Expression of the global neddylation protein, NEDD8activating enzyme E1 and NEDD8-conjugating enzyme E2 in ESCC tissues. A, IB
analysis to determine the expression of NEDD8 conjugated protein in ECSS tissues
and adjacent normal tissues. Representative results of 4 of 10 pairs of tissues were
shown (left panels). Protein expression was quantified and analyzed (right panels)
(Error bar = S.D.). B, IHC staining of human ESCC tissue arrays using NAE1, UBA3
and UBC12-specific antibodies. According to the staining intensity of NAE1, UBA3
and UBC12, tumor samples were classified into five groups from the weakest (±,
group 1) to the strongest (++++, group 5) according to the description in Figure 1. C,
Classification of tumor samples according to the staining intensity of NAE1, UBA3
and UBC12 (n=92). Statistical analyses of staining intensity by Mann-Whitney Test
showed that there are significant difference between tumor and their adjunct tissues
(P<0.01). D, IB analysis to determine the expression of NAE1, UBA3, and UBC12 in
ECSS tissues and adjacent esophageal tissues. Representative results of 4 out of 10
pairs of tissues were shown (upper panel). Quantification of proteins expression in
ESCC tissues compared with adjacent esophageal tissues. The results of a total 10
pairs of tissues (tumor vs adjacent normal tissues) were analyzed (lower panel).
Horizontal lines indicated means ± standard deviation (Error bar = S.D.). Two-sided t
test. A = adjacent normal tissues; T = tumor tissues.
Supplementary Figure S2. Expression of death-receptor family and Bcl-2 family
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members. Kyse450 and EC1 cells were treated with indicated concentration of
MLN4924 for 72 hours. Cells were subjected for IB analysis to measure the
expression of death-receptor family members (A) and Bcl-2 family members (B) with
the indicated antibodies. GAPDH served as a loading control.
Supplementary
Figure
S3.
MLN4924
acted
on
DR5-mediated
apoptosis
independently of Trail. Kyse450 and EC1 cells were transfected (48 hours) with
control or Trail siRNA, treated with 0.6 μM MLN4924 (48 hours). Apoptosis was
quantified by Annexin V-FITC/PI double-staining analysis (A) or CASP3 activity
analysis with FACS (B). Knockdown efficiency and cleaved PARP were assessed by
IB analysis (C). All data were representative of at least three independent experiments
(Error bar = S.D.).
Supplementary Figure S4. MLN4924 inhibited the polyubiquitination modification of
ATF4. A, MLN4924 increased the mRNA level of CHOP while little effect on ATF4
mRNA level. Kyse450 and EC1 cells were treated with MLN4924 (0.6 μM) at the
indicated time. RNA extracts were prepared, and the mRNA level of ATF4 and CHOP
was determined by the qPCR assay. B, MG-132 enhanced the protein stability of
ATF4. Kyse450 and EC1 cells were pretreated with MG-132(10 μM) for 6 hours to
increase the basal protein level of ATF4. Cells were then washed with PBS to remove
residual drug and divided into 2 groups, which were further treated with CHX (50
μg/ml) +DMSO or CHX (50 μg/ml) +MG-132 (10 μM) for indicated times and then
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collected for IB analysis. C, MLN4924 inhibited the polyubiquitination modification
of ATF4. Kyse450 and EC1 cells were pretreated with MG132 and starved for 12
hours, and then cells were treated with MLN4924, along with DMSO control for
another 12 hours, followed by serum addition for 2 hours. Cells were extracted and
subjected to IP with anti-ATF4 Ab and IB with anti-ubiquitin Ab.
Supplementary Figure S5. A, Knockdown efficiency of ATF4. Kyse450 and EC1 cells
were transfected with control or ATF4 siRNA for 24 hours and then treated with 0.6
μM MLN4924 for 48 hours. Knockdown efficiency was assessed by qPCR according
to Material and Methods. B, Knockdown efficiency of CHOP. Knockdown efficiency
of CHOP was evaluated as descripted for ATF4 in panel A. C, Accumulation of ATF4,
CHOP, DR5 and Noxa in MLN4924-treated ESCC cell lines. EC109, Kyse30 and
Kyse510 cells were treated with MLN4924 for 72 hours and cell lysates were
assessed by IB with specific antibodies against ATF4, CHOP, DR5 and Noxa.
GAPDH served as a loading control.
Supplementary Figure S6.
Genetic inactivation of neddylation pathway via
NAE1/UBA3 siRNA silencing recapitulates MLN4924-induced cellular responses in
ESCC cells. Knockdown of UBA3 and NAE1 impaired the cell viability of Kyse450
and EC1 cells. Cells were transfected with control siRNA, UBA3 or NAE1 siRNA for
96 hours, and then cell viability was assessed with the ATPLite assay (A) and
apoptosis was determined by Annexin V-FITC/PI double-staining analysis (B), cell
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morphology were recorded (C), cell lysates were collected and subjected to IB
analysis with the indicated antibodies (D). GAPDH served as a loading control.
Supplementary Figure S7. No body weight loss occurred during treatment. Body
weight of the animals was measured and recorded during the treatment period.
Supplementary Table S1. Clinicopathologic parameters according to the expression of
NAE1, UBA3 and UBC12.
Supplementary Table S2. Clinicopathologic parameters according to the expression of
global neddylation (global NEDD 8 conjugation).
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