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Figure S1
A
B
Firefly-Luc
Firefly-Luc
c-Myc IRES Renilla-Luc
Renilla-Luc
c-Myc
USP37
Renilla-Luc
Stabilization
Figure S1. (A) Screen of USPs for regulating c-Myc stability. Scores of c-Myc protein abundence was
derived from two independent experiments. Plotted c-Myc bands intensities were quantified by Odyssey V3.0
software, normalized to Actin protein level. The scores of control empty vector were assigned as 1. c-Myc
expression cotransfected with USPs are shown. RPL, relative c-Myc protein level. (B) Measure the
stabilization of c-Myc by USP37 using luciferase based protein degradation system (LPDS). Construction of
LPDS was indicated. The LPDS-c-Myc was expressed in 293T cells for 36 hours. Transfected cells were lysed.
The luciferase activity was measured by Luciferase reporter System (Promega). The protein abundance was
determined by the ration of Firefly luciferase/Renilla luciferase.
Figure S2
A
HA-c-Myc +
Flag-Fbw7α Flag-USP37 -
+
+
-
+
+
+
+
+
HA-c-Myc
Flag-Fbw7α
Flag-USP37
EGFP
B
Myc WT T58A S62A T58/S62A
Flag-USP37 -
+ -
+ -
+ -
+
Myc/AA mutant
Flag-USP37
EGFP
C
siNC
siUSP37 -
siFbw7
+
-
+
Myc
USP37
Actin
Figure S2.The regulation of c-Myc by USP37 is independent of Fbw7. (A) The degradation of c-Myc by
Fbw7 is blocked by USP37. 293T cells were transfected and protein levels were monitored by
immunoblotting. (B) USP37 were co-expressed with wildtype c-Myc, c-MycT58A, c-MycS62A or c-MycT58/S62A in
293T cells and related proteins were detected using Western blotting. (C) Cells were transfected with siRNA
against USP37 or Fbw7 as indicated. The protein levels were analyzed using Western blotting.
Figure S3
A
B
HA-MYC Flag-USP37
MERGE
GFP-USP37
HA-MAX
Flag-c-Myc
+ + + +
- + - +
- - + +
IB:GFP
IP:HA
IB:HA
IB:Flag
WCE
IB:HA
IB:Flag
IB:GFP
Figure S3. Colocalization of USP37 and c-Myc in the nucleus. (A) HA-c-Myc and Flag-USP37 were coexpressed into HeLa cells. Transfected cells were treated MG132, fixed, and the USP37 was stained with an antiFlag antibody while c-Myc with anti-HA antibody. Images were analyzed by confocal microscopy. (B) HA-MAX or
Flag-c-Myc were coexpressed with GFP-USP37 in HEK293T cells. Transfected cells were treated with MG132,
and the cell lysates were immunoprecipitated using an anti-HA antibody. Co-immunoprecipitated USP37 was
detected using an anti GFP-antibody.
Figure S4
A
Rs=0.24
P=6.6e-08
n=490
5000
2500
0
0
1000
2000
USP37 expression (RNA seq)
CCNB1 expression (RNA seq)
B
BRCA
3000
2000
1000
0
Normal
n=110
USP37 expression (RNA seq)
NS
1000
500
0
Normal
n=50
HNSC
1500
NS
2000
1500
1000
500
0
Normal
n=67
Tumour
n=428
NS
500
0
Normal
n=37
KIRC
2500
Tumour
n=1044
1000
Tumour
n=141
USP37 expression (RNA seq)
USP37 expression (RNA seq)
USP37 expression (RNA seq)
USP37 expression (RNA seq)
PRAD
***
Tumour
n=303
COAD
2000
*
1500
1000
500
0
Normal
n=41
Tumour
n=455
Figure S4. Expression of USP37 in cancers. (A) The expression of USP37 is correlated with CCNB1.
Scatter plots show a positive and significant correlation of mRNA expression between USP37 and the c-Myc
target gene CCNB1 in lung adenocarcinoma. (B) mRNA expression data sets showing increased USP37
mRNA levels in breast invasive carcinoma(BRCA),but not in prostate adenocarcinoma(PRAD),head and neck
squamous cell carcinoma(HNSC), kidney renal clear cell carcinoma(KIRC) or colon adenocarcinoma (COAD)
compared to normal tissue.
Figure S5
B
A
USP37
USP37
DAPI
MERGE
Antibody
absorbed
siUSP37
siNC
Control
Figure S5. Specificity of USP37 antibody. (A) H1299 cells were transfected with control or USP37 siRNA.
USP37 was examined using immunofluorescence assay. (B) 0.4 mg USP37 antibody was incubated with 40 mg
purified His-USP37 protein for 4 hours. Serial sections of lung cancer samples were stained with control antibody
and the antibody absorbed by USP37 protein.