Supplementary Fig. S1 A.B.. A. Supplementary Fig. S2 B. C.
9
Supplementary Fig. S TU M 622 M D A 435-5T4 M D A-468 M D A-361-D YT2 CaO V-3 TO V-112D H 1975 Raji 5T4 (EPR 5530) GAPDH D L T U M 6 22 M D A 435 /5T4 MDA4 6 8 M D A 36 1 D YT2 C aov3 T OV 1 1 2D H 1 9 7 5 R a j i 0 50000 100000 250000 5T4 A ntibody B inding C apacity 5T4 A 1 B inding sites /C ell A. B.
-
Upload
felix-marsh -
Category
Documents
-
view
227 -
download
3
Transcript of Supplementary Fig. S1 A.B.. A. Supplementary Fig. S2 B. C.
Supplementary Fig. S1
TU
M62
2
MD
A43
5-5
T4
MD
A-4
68
MD
A-3
61-
DY
T2
CaO
V-3
TO
V-1
12D
H19
75
Raj
i5T4
(EPR5530)
GAPDH
D
L
TUM62
2
MDA43
5/5T
4
MDA46
8
MDA36
1DYT2
Caov3
TOV11
2D
H1975 Raj
i0
50000
100000
250000
5T4 Antibody Binding Capacity
5T
4 A
1 B
ind
ing
sit
es
/ C
ell
A. B.
PF384 (50nM)
Control
MMAF-Ome+ PF384
MMAF-Ome(0.06 nM)
A. Supplementary Fig. S2
B.
C.
MDA-468, 8 Days
0.0
0.5
1.0
1.5
2.0
Imp
ed
an
ce
Ind
ex
Control PF-384 MMAF-OMe MMAF-OMe+ PF-384
*******
MDA-468, Spheroid Growth, 7 Days
0.1 1 10 100 10000
25
50
75
100
125
PF-384 (nM)
Sp
he
roid
Via
bil
ity
(%
co
ntr
ol) PF-384
PF-384+MMAF-OMe (0.8)
MMAF(0.8)
0
2
4
6
8
MDA468, 24h
PF-384MMAF-OMe MMAF-OMe+PF-384
Fo
ld In
du
cti
on
(C
as
pas
e 3
/7)
***
*
0
2
4
6
H-1975, 24h
PF384MMAF-OMe MMAF-OMe+PF384
Fo
ld In
du
cti
on
(C
as
pas
e 3
/7)
*
**
0
20
40
60
80
100
MDA-468, Spheroid Growth, 7 Days
Sp
her
oid
Via
bil
ity
(% c
on
tro
l)
PF-384(25 nM)
MMAF-OMe(0.8 nM)
MMAF-OMe+PF-384
*****
A. Supplementary Fig. S3
C.B.
Cntr
PF384
(100
nM)
5T4-
ADC (1g
)
PF384
(100
nM) +
5T4-
ADC (1g
) Cntr
PF384
(100
nM)
5T4-
ADC (1g
)
PF384
(100
nM) +
5T4-
ADC (1g
)
0
5
10
15Mitotic Cells MDA468
% P
-His
ton
e H
324 hr
36 hr
Cntr
PF384
(100
nM)
5T4-
ADC (10
g)
PF384
(100
nM) +
5T4-
ADC (10
g) Cntr
PF384 (
100n
M)
5T4-
ADC (10
g)
PF384
(100
nM) +
5T4-
ADC (10
g)
0
5
10
15
20Mitotic Cells MDA468
% P
-His
ton
e H
3
24 hr
36 hr
Control PF384(100nM)
5T4-ADC(10g)
PF384/5T4-ADC
0
20
40
60
Cell Cycle MDA468, 24h
% C
ells
sub G1
G1
G2
S
Control PF384(100nM)
5T4-ADC(10g)
PF384/5T4-ADC
0
20
40
60
Cell Cycle MDA468, 36h
% C
ell
s
sub G1
G1
G2
S
Cntr
PF-384
(100
nM)
MM
AF-Om
e
(0.1
nM) M
MAF
(0.5
nM)
PF-384
(100
nM)
+ M
MAF (0
.1nM
)
PF-384
(100
nM)
+ M
MAF (0
.5nM
)
0
5
10
15
20
25
Mitotic Cells H-1975, 24h
% P
--H
isto
ne
H3
D.
P-Aurora A/B/C
Vinculin
- 10 50 - - 10 10 50 50
- - - 5 50 5 50 5 50
16h 24h
- 10 50 - - 10 10 50 50 5T4-ADC, g/ml
- - - 5 50 5 50 5 50 PF-384, nM
H-1975
Aurora B
Supplementary Fig. S4
A. B.
P-AKT1S473
P-S6KT389
P-S6KT421/S424
P-GSK3S9
P-eNOSS1177
P-PRAS40T246
0.0
0.2
0.4
0.6
0.8
1.0
1.2
H-1975, PF-384
Pix
el D
en
sit
y F
old
(/
Cn
trl)
6 hr
16 hr
P-AKT1S473
P-PRAS40T246
P-GSK3S9
0.0
0.2
0.4
0.6
0.8
1.0
H-1975, 5T4-ADC 16h
Pix
el D
en
sit
y F
old
(/
Cn
trl)
P-S6K T421/S424
P-GSK3 S9
P-eNOS S1177
P-PRAS40 T246
P-S6K T389
P-AKT1
P-AKT2
P-AKT3
P-pan-AKT
P-GSK3 S9
P-PRAS40 T246
P-AKT1 S473
MDA-468, 24h
- 5 50 - - 5 50 5 50 PF-384, nM
- - - 1 10 1 1 10 10 5T4-ADC, g/ml
P-AKT (T308)
P-AKT (S473)
P-GSK (S21/9)
GAPDH
cPARP
GSK
AKT
P-H3 (S28)
1 2 3 4 5 6 7 8 9
1 2 3 4 5 6 7 8 90.00
0.25
0.50
0.75
1.00
Re
lati
ve
De
ns
ito
me
tric
In
de
x
P-AKT473
P-AKT308
1 2 3 4 5 6 7 8 90.00
0.25
0.50
0.75
1.00
1.25
Re
lati
ve
De
ns
ito
me
tric
In
de
x
P-GSKP-GSK
C.
MMAF-OMe (0.5nM)
DownregulatedDAP3, RBM3, EEF2, EIF3C, EIF3CL, EIF5, EIF5A, HARS, MRPL13, MRPL16, MRPS15, NACA, RPL23, EIF4A2, EIF4A1
Upregulated
FAU, TUFM, RARS2, EEF1D, MRPL15, MRPL18, MRPL24, MRPS16, PTRH2, RPL13, RPL14, RPL18, RPL18A, RPL21P14, RPL22, RPL34, RPL35, RPL36A, RPL36AL, RPS13, RPS14, RPS19, RPS27A, RPS3A
MMAF-Ome (5nM)
DownregulatedDAP3, RBM3, EEF2, EIF5A, MRPL13, MRPL16, MRPL41, MRPL43, PA2G4, RPS21, EIF4A1
Upregulated
FAU, RARS2, EIF3L, MRPL15, MRPL18, MRPL3, MRPL9, MRPS7, RPL13, RPL13A, RPL18, RPL18A, RPL19, RPL21P14, RPL24, RPL26, RPL27, RPL30, RPL31, RPL34, RPL35, RPL36A, RPL36AL, RPS13, RPS14, RPS15A, RPS16, RPS26, RPS27A, RPS3A, RPLP0, RPLP1, RPLP2, RPL32
Supplementary Fig. S5
D.MMAF-Ome (1 nM)
Downregulated
RPLP2 (S102, S105), EIF2S2 (S105, T111), RPLP0 (S304, S307), EIF4ENIF1 (S74, S77), EIF4EBP1 (S65, T68, T70), EIF5B (S134, S135, S137), EEF1B2 (S106), EEF1D (S133), TARBP2 (S152), UPF1 (S1111, S1112), PUM1 (S124)
UpregulatedRSL1D1 (T423, S427), RPS6 (S235, S236, S240), eEF2 (T54, T57, T59), AKT (S124)
5T4ADC (1 g/ml)
Downregulated
TARBP2 (S152), EIF4G1 (S1596), EIF4EBP1 (S65, T69, T70), EIF4ENIF1 (S74, S77), EIF2S2 (S105, T111), EIF5 (S389, S390), EIF5B (S134, S135, S137), RPLP2 (S102, S105), EEF1B2 (S106), NGDN (S142, S143), RPLP0 (S304, S307), EEF1D (S133)
UpregulatedRPS6 (S235, S236, S240), EIF5B (S113, S107), eEF2 (T59)
B.
MMAF-OMe (0.5 nM) MMAF-OMe (5 nM)
Decreased
Increased
Total Proteomics
MMAF-OMe 5T4-ADC
Decreased
Increased
Phosphoproteomics
C. E.
A.
Supplementary Fig. S6
A.
Control
PTX (1nM
)
5T4-
ADC (1g
/ml)
PTX-1 /
5T4
ADCContro
l
PTX (10n
M)
5T4-
ADC (1g
/ml)
PTX-10
/
5T4
ADC
0
2
4
6
8Mitotic Cells MDA-468, 24h
% P
-His
ton
e H
3
Control
PTX (1nM
)
5T4-
ADC (10
g/ml)
PTX-1 /
5T4
ADCContro
l
PTX (10n
M)
5T4-
ADC (10
g/ml)
PTX-10
/
5T4
ADC
0
5
10
15
20Mitotic Cells, MDA-468 24h
% P
-His
ton
e H
3
Control 5T4-ADC
PTX 5T4-ADC/PTX
MDA-468
MMAF-OMe
MMAF-Ome/PTX
B.
Cell Lines Type Mutations
TUM622 NSCLC, AA37622 PDX-derived K-RAS+/-
NCI-H1975 NSCLC, Adenocarcinoma EGFR+/- (L858R/T790M), PIK3CA+/-(G118D), TP53-/-, CDKN2A-/-
Calu-6 NSCLC TP53-/-, KRAS+/-, BRCA2+/-
NCI-H358 NSCLC, Bronchioloalveolar adenocarcinoma KRAS+/-, FGFR1+/-
HCC2429 NSCLC N.A.
MDA-MB-435-5T4 Melanoma, pleural effusion BRAF+/-(V600E), TP53+/-, CDKN2A+/-
MDA-MB-468 Breast, Basal HER2-/ER2-/PrgR-; PTEN-/-, RB1del-/-, SMAD4-/-, TP53-/-
MDAMB-361-DYT2 Breast, Luminal HER2+/ER2+/PrgR-; 2XPIK3CA+/-, AKT1+/-, EGFR+/-
MDA-231 Breast, Basal HER2+/ER-/PrgR-; K-RAS+/-, B-RAF+/-, NF2-/-. TP53-/-, CDKN2A-/-
CAOV-3 Ovarian, Adenocarcinoma TP53-/-, EGFR+/-(R255Q), STK11-/-
TOV-112DOvarian, malignant adenocarcinoma; endometrioid carcinoma
TP53-/-, HER2+
OV-90 Ovarian, Malignant Papillary Serous Adenocarcinoma TP53-/-, SMAD4-/-. BRAF+/-, CDKN2A-/-
OVCAR-3 Ovarian, Adenocarcinoma TP53-/-, PPP2R1A+/-
SKOV-3 Ovarian, Adenocarcinoma PIK3CA+/-(H1047R), TP53-/-, NF1+/-, ATM+/-, APC+/-
HT-29 Colorectal adenocarcinoma BRAF+/-(V600E), PIK3CA+/-(P449T), SMAD4-/-, TP53-/-, 2XAPC+/-
NCI-N87Gastric carcinoma, Adenocarcinoma, derived from metastatic site, liver
SMAD4-/-, TP53-/-, HER2+/-, IL2+/-, PCSK7+/-
Raji NHL MYC+/-, TP53+/-
Supplementary Table S1
Total Proteomics, DAVID
Condition Change GO Category Count P Value
MMAF-Ome (0.5 nM) Decreased GOTERM_BP_ALL translation 145.6E-5
MMAF-Ome (5 nM) Decreased GOTERM_BP_ALL translation 10 1.1E-3
MMAF-Ome (0.5 nM) Increased GOTERM_BP_ALL translation 24 6.3E-14
MMAF-Ome (5 nM) Increased GOTERM_BP_ALL translation 34 2.5E-22
Phospho Proteomics, DAVID
Condition Change GO Category Count P Value
MMAF-Ome1 nM
Decreased GOTERM_BP_ALL
ribonucleoprotein complex biogenesis
9 8.7E-4
ribosome biogenesis 6 1.1E-2
Regulation of translation 6 1.8E-2
5T4-ADC3 g/ml
Decreased GOTERM_BP_ALL Regulation of translation 10 8.3E-4
MMAF-Ome1 nM
Increased GOTERM_BP_ALL RNA metabolic process 20 1.2E-4
5T4-ADC3 g/ml
Increased GOTERM_BP_ALLRegulation of translation 5 3.9E-2
mRNA metabolic process 16 1.7E-6
Supplementary Table S2A
Supplementary Table S2B
Fixed Drug Ratios CI Index at ED50
Aur101 : WYE-132 HT-29 N-87
1:2.7 0.18 (0.09) 0.04 (0.02)
1:8.3 0.39 (0.04) 0.35 (0.04)
1:25 0.43 (0.04) 0.53 (0.28)
1:75 0.58 (0.02) 0.52 (0.12)
1:225 0.87 (0.21) 0.49 (0.16)
1:675 0.57 (0.05) 0.41 (0.09)
1:2025 0.43 (0.07) 0.40 (0.13)
Supplementary Table S3
![Supplementary Fig. 1. Plasmids luciferase …...Supplementary Fig. 4. Study flowchart. Study flowchart providing a framework of cases [samples] inclusion from patients series (Initial](https://static.fdocuments.us/doc/165x107/5e30bee5761fd5400c33deb9/supplementary-fig-1-plasmids-luciferase-supplementary-fig-4-study-flowchart.jpg)
