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1

Supplementary Information

Peptidomimetic inhibitors of APC-Asef interaction block

colorectal cancer migration

Haiming Jiang1,||

, Rong Deng1,2,||

, Xiuyan Yang1,||

, Jialin Shang1, Shaoyong Lu

1,

Yanlong Zhao1, Kun Song

1, Xinyi Liu

1,3, Qiufen Zhang

1,3, Yu Chen

4, Y. Eugene

Chinn5, Geng Wu

6, Jian Li

7, Guoqiang Chen

1,5, Jianxiu Yu

1,2,* Jian Zhang

1,2,3,*

1Department of Pathophysiology, Key Laboratory of Cell Differentiation and

Apoptosis of Ministry of Education, Shanghai Jiao-Tong University School of

Medicine, Shanghai 200025, China.

2Department of Biochemistry and Molecular Cell Biology, State Key Laboratory of

Oncogenes and Related Genes, Shanghai Key Laboratory of Tumor

Microenvironment and Inflammation, Shanghai Jiao-Tong University School of


3Medicinal Bioinformatics Center, Shanghai Jiao-Tong University School of


4State Key Laboratory of High Performance Ceramics and Superfine Microstructure,

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050,

China.

5Institute of Health Sciences, Shanghai Institutes for Biological Sciences of Chinese

Academy of Sciences and Shanghai Jiao-Tong University School of Medicine,

Shanghai 200031, China.

Nature Chemical Biology: doi:10.1038/nchembio.2442

2

6State Key Laboratory of Microbial Metabolism, Shanghai Jiao-Tong University,

Shanghai 200240, China.

7Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China

University of Science and Technology, 130 Meilong Road, Shanghai 200237, China.

|| The authors equally contribute to this work.

*To whom correspondence should be addressed:

Dr. Jian Zhang, PhD

Phone: +86-21-63846590-776922

Fax: +86-21-64154900

E-mail: [email protected]

Dr. Jianxiu Yu, PhD

Phone: +86-21-63846590

Fax: +86-21-64154900

E-mail: [email protected]


3

Supplementary Results

Supplementary Tables

Supplementary Table 1. Sequences and activities of representative peptide inhibitors

for binding to APC. a

ID SEQUENCE KiSEM(μM)b IC50SEM(μM)

ORIGIN Ac-SHPGGGGEQLAINELISDG-NH2 16.720.28 50.950.85

MAI-001 Ac-GGGGEQLAINELISDG-NH2 17.103.52 52.0610.58

MAI-002 Ac-GGGEQLAINELISDG-NH2 19.512.88 59.287.09

MAI-003 Ac-GGEQLAINELISD-NH2 49.113.29 147.9020.5

MAI-004 Ac-GGEQLAIN-NH2 52.076.87 156.9520.62

MAI-005 Ac-GGEQLAI-NH2 44.620.99 134.602.97

MAI-006 Ac-GGEQLA-NH2 81.417.51 24555.54

MAI-007 Ac-GGEQL-NH2 300 1000

MAI-008 Ac-GEQLAIN-NH2 73.713.72 221.9033.60

MAI-009 Ac-EQLAINEL-NH2 300 1000

MAI-010 Ac-EQLAINE-NH2 300 1000

MAI-011 Ac-LAINEL-NH2 300 1000

MAI-012 Ac-INELISD-NH2 300 1000

MAI-013 Ac-GEQLAI-NH2 138.741.93 4174.74

MAI-014 Ac-GEQLA-NH2 300 1000

MAI-015 Ac-GGAQAA-NH2 300 1000

MAI-016 Ac-GGEQAA-NH2 300 1000

MAI-017 Ac-GGAQLA-NH2 300 1000

MAI-018 Ac-AGEQLAI-NH2 67.6126.36 203.6079.08

MAI-019 Ac-TGEQLAI-NH2 300 1000

MAI-020 Ac-DGEQLAI-NH2 300 1000

MAI-021 Ac-GVEQLAI-NH2 300 1000

MAI-100 Ac-GGEPLAI-NH2 300 1000

MAI-022 Ac-GGEQLII-NH2 300 1000

MAI-023 Ac-GGEQLLI-NH2 300 1000

MAI-024 Ac-GGEQLAL-NH2 43.3511.48 130.8034.43

MAI-025 Ac-GGEQLAY-NH2 44.458.10 134.1024.32

MAI-026 Ac-GGEQLAD-NH2 10.451.06 32.103.18

MAI-027 Ac-GGEQLAW-NH2 12.921.20 39.503.60

MAI-028 Ac-GGESLAI-NH2 5.730.43 17.931.30


4

MAI-029 Ac-GGEALAI-NH2 4.051.39 12.904.17

MAI-030 Ac-GGEALSD-NH2 11.011.72 33.795.15

MAI-031 Ac-GGEALTD-NH2 69.258.04 208.5024.11

MAI-032 Ac-GGEALVD-NH2 36.422.06 1106.18

MAI-033 Ac-GGEALDD-NH2 15.851.53 48.314.60

MAI-034 Ac-GGEALAA-NH2 322.13123.05 967.2369.17

MAI-035 Ac-GYEALAD-NH2 74.182.77 223.308.30

MAI-036 Ac-GGEANAD-NH2 381.4049.43 1145148.30

MAI-037 Ac-AGEALAW-NH2 3.900.29 12.460.88

MAI-038 Ac-GGEALA-NH2 25.802.69 78.158.05

MAI-039 Ac-GGEANA-NH2 364.068.00 109324.00

MAI-040 Ac-GEALA-NH2 19.391.37 58.914.11

MAI-041 Ac-GEAL-NH2 652.7178.53 1959235.61

MAI-100 Ac-GGEPLAI-NH2 300 1000

MAI-101 Ac-GGEALYS-NH2 1.440.29 5.050.88

MAI-102 Ac-GGEALAW-NH2 3.120.70 10.122.10

MAI-103 Ac-GGEALYE-NH2 0.620.15 2.600.47

MAI-104 Ac-GGEALY-NH2 7.112.14 22.076.42

MAI-105 Ac-AGEALYE-NH2 0.320.08 1.700.25

MAI-106 Ac-AGEALYD-NH2 0.990.12 3.710.34

MAI-107 Ac-GGEALAD-NH2 3.800.72 12.162.16

MAI-108 Ac-AGEALAD-NH2 2.410.88 7.972.65

MAI-109 Ac-AGDALYE-NH2 1.190.13 4.310.38

MAI-110 Ac-AGEALFE-NH2 1.070.19 3.960.55

MAI-111 Ac-AGEALWE-NH2 1.810.07 6.160.22

MAI-112 Ac-AGEALYQ-NH2 1.800.08 6.130.25

MAI-113 Ac-SGEALYE-NH2 1.430.20 5.030.61

MAI-114 Ac-SGDALYE-NH2 4.160.34 13.231.01

MAI-115 Ac-GYEGYYS-NH2 303.409.68 91129.04

MAI-116 Ac-GGDALYE-NH2 8.790.78 27.112.32

MAI-117 Ac-GGEALYD-NH2 1.500.06 5.250.18

MAI-118 Fmoc-AGEALYE-NH2 4.020.48 12.811.45

MAI-119 Z-AGETLYE-NH2 0.4120.067 1.9810.20

MAI-120 Z-AGEDLYE-NH2 1.640.24 5.670.72

MAI-121 Z-AGEAHYE-NH2 300 1000

MAI-122 Z-AGESLYQ-NH2 0.0760.006 0.97310.16

MAI-123 Z-LGYEAEA-NH2 1000 3000


5

MAI-150 Z-AGEALYE-NH2 0.120.02 1.090.06

aEffects of test peptide inhibitors on the fluorescence polarization (FP) competitive assay for APC were

assessed as described in the Materials and Methods. bKi and IC50 values shown are the average of three

independent experiments in triplicate with typical variations of less than 20%. Z, benzyloxycarbonyl;

Fmoc, fluorenylmethoxycarbonyl; Ac, N-terminally acetylated; NH2, C-terminally amidated.


6

Supplementary Table 2. Data collection and refinement statistics.*

APC/

MAI-102

APC/

MAI-107

APC/

MAI-108

APC/

MAI-150

APC/

MAI-203

Data collection Space group P212121 P212121 P6522 P21 P21 Cell dimensions a, b, c (Å) 52.465,

66.436,

82.661

64.133,

64.559,

84.912

114.073,

114.073,

308.295

51.396,

63.215,

52.768

51.448,

63.691,

52.641 () 90.00, 90.00,

90.00

90.00, 90.00,

90.00

90.00, 90.00,

120.00

90.00, 95.61,

90.00

90.00,

95.763,

90.00 Resolution (Å) 50-1.50

(1.55-1.50)

50-2.91

(3.01-2.91)

50-3.27

(3.27-3.06)

50-2.15

(2.2-2.15)

50-1.99

(2.06-1.99) Rsym or Rmerge 0.064 (0.545) 0.124 (0.605) 0.273 (0.815) 0.096 (0.473) 0.068 (0.249) I / I 21.3 (2.9) 12.5 (3.5) 16.2 (5.6) 11.2 (2.0) 15.2 (5.0) Completeness (%) 99.5(99.9) 99.1(99.9) 99.9 (99.8) 99.4 (97.3) 99.2 (99.6) Redundancy 4.7 (4.7) 4.4(4.6) 21.1 (21.5) 3.0(2.6) 3.3(3.3) Refinement Resolution (Å) 1.50 2.93 3.06 2.15 1.99 No. reflections 220085 34671 495864 55513 75983 Rwork / Rfree 0.121/0.159 0.233/0.282 0.213/0.253 0.163/0.206 0.170/0.215 No. atoms 3296 2600 5373 2903 2969 Protein 2896 2543 5161 2627 2651 Ligand/ion 53 47 96 63 65 Water 347 10 106 201 242 B-factors 24.963 79.749 60.241 31.469 27.438 Protein 23.353 80.040 61.181 30.772 26.493 Ligand/ion 24.847 69.787 44.338 39.879 41.385 Water 38.418 52.463 28.380 36.245 32.987 R.m.s. deviations Bond lengths (Å) 0.011 0.007 0.01 0.01 0.01

Bond angles () 1.452 1.169 1.398 1.363 1.514

*Highest-resolution shell is shown in parentheses. Data were collected from one crystal for each

structure.


7

Supplementary Table 3. Thermodynamic parameters of peptide inhibitors for

binding to APC, APC mutants, and Asef determined by isothermal titration

calorimetry (ITC).*

Ligand Receptor L/P ratio H (kcal/mol) TS (kcal/mol) Kd (μM)

MAI-150 APC 0.9 10.30 1.10 0.23

MAI-150 Asef N/A N/A N/A N/A

MAI-150 APC-D539A 1.01 11.87 2.86 0.32

MAI-150 APC-R549A 1.05 16.37 10.06 28.01

MAI-150 APC-K586A 1.07 11.11 2.67 0.83

MAI-150 APC-W593A 1.03 10.54 3.55 9.35

MAI-150 APC-K516A N/A N/A N/A N/A

MAI-150 APC-N507W N/A N/A N/A N/A

MAI-100 APC N/A N/A N/A N/A

MAI-201 APC 0.907 12.53 3.39 0.26

MAI-202 APC 1.14 8.14 0.62 0.49

MAI-203 APC 1.08 11.19 0.87 0.036

MAI-204 APC 0.87 9.88 0.29 0.12

*All experiments were performed at 30°C. N indicates number of sites per APC. N/A indicates that no

detectable interaction was observed. H: change in enthalpy; TS: change in entropy; Kd: Equilibrium

dissociation constant determined by ITC.


8

Supplementary Table 4. Structures and binding affinities of representative MAI-150

derivatives.a

ID R1 R2 R3 R4 R5 KiSEM(μM)

MAI-150 H H

H OH 0.120.02

MAI-151 H H

H F 0.850.06

MAI-152 H H

H NO2 2.860.22

MAI-153 H H

H NH2 0.5940.06

MAI-154 H H

Cl H 0.420.005

MAI-155 H H

I H 0.370.016

MAI-201 H H

H OH 0.060.001*

MAI-202 H H

H OH 0.3420.06

MAI-203 H H

H OH 0.0150.0001*

MAI-204 H H

H OH 0.0160.0001*

MAI-205 H H

H OH 0.6250.09

MAI-206 H H

H OH 0.8150.24

MAI-207 F H

H OH 0.1570.03


9

MAI-208 H OH

H OH 0.1460.01

MAI-209 H F

H OH 0.4560.035

a Ki and IC50 values shown are the average of three independent experiments in triplicate with typical

variations of less than 20%. * indicates that the test values were below the theoretical limit and the Ki

values were estimated based on the original equation.


10

Supplementary Table 5. Thermal stability of representative MAI inhibitors for

binding to APC.a

ID Ki (μM) Tm (С, MeanSD)

b

MAI-protein

Tm (С, MeanSD)c

Apo protein

∆Tm (С, MeanSD)d

MAI-203 0.015 50.970.004 43.950.002 7.020.002

MAI-150 0.12 50.000.001 43.950.002 6.050.001

MAI-108 2.41 48.010.002 43.950.002 4.060.000

MAI-107 3.75 47.970.004 43.950.002 4.020.002

MAI-102 3.12 47.050.003 43.950.002 3.100.001

MAI-005 44.62 44.950.002 43.950.002 1.000.000

MAI-100 300 44.020.002 43.950.002 0.070.000

a Tm (Temperature degree, С) values are the mean of three independent experiments based on the same

batch of APC and peptides. The peptides that bind to APC induce thermal stabilization of the protein,

as reflected by an increase in Tm. b The Tm of APC mixed with 250 µM peptide.

c The Tm of APC mixed

with DMSO. d

ΔTm was calculated as the difference between the protein mixed with 250 µM peptide

and the protein mixed with DMSO. The Ki values determined by FP are also reported for peptide

inhibitors. MAI-005, MAI-102, MAI-107, MAI-108, MAI-150, and MAI-203 bind to APC and

increase Tm. No protein stabilization is observed for negative control peptide, MAI-100.


11

Supplementary Table 6. Binding activities of the TAT-derived inhibitors optimized

based on MAI-150.a

SEQUENCE KiSEMb

(μM)

IC50SEM

(μM)

Ac-GRKKRRQRRRGGAGEALYE-NH2 41.320.08 124.70.25

Ac-GRKKRRQRRRGGGAGEALYE-NH2 64.486.95 194.220.85

Ac-GRKKRRQRRRGGGGAGEALYE-NH2 34.021.36 102.84.05

Ac-GRKKRRQRRRGGGGGAGEALYE-NH2 16.472.52 50.177.55

Ac-GRKKRRQRRRGGGGGGAGEALYE-NH2 8.250.47 25.51.41

Ac-GRKKRRQRRRGGGGGGGGAGEALYE-NH2 5.260.43 16.541.30

Ac-GRKKRRQRRRGGGGGGGGGAGEALYE-NH2 4.730.67 14.932.00

Z-AGEALYEGGRKKRRQRRR-NH2 6.700.45 20.841.35

Z-AGEALYEGGGRKKRRQRRR-NH2 4.991.09 15.723.29

Z-AGEALYEGGGGRKKRRQRRR-NH2 3.080.18 9.980.54

Z-AGEALYEGGGGGRKKRRQRRR-NH2(MAIT-150) 1.460.35 5.121.04

Z-AGEALYEGGGGGGRKKRRQRRR-NH2 1.780.17 6.070.50

Z-AGEALYEGGGGGGGRKKRRQRRR-NH2 1.650.02 5.690.07

Z-AGEALYEGGGGGGGGRKKRRQRRR-NH2 1.360.09 4.830.27

Z-AGEALYEGGGGGGGGGRKKRRQRRR-NH2 1.490.12 5.220.34

Z-AGEA(s-3cyclopentylalamine)YEGGGGGRKKRRQRRR-NH2 (MAIT-203) 0.400.092 1.9420.28

Ac-GGEPLAIGGGGGRKKRRQRRR-NH2 (MAIT-100) 300 1000

a Effects of test peptide inhibitors on the fluorescence polarization (FP) competitive assay of APC were

assessed as described in the Materials and Methods. b Ki and IC50 values shown are the average of three

independent experiments in triplicate with typical variations of less than 20%. Z, benzyloxycarbonyl;

Ac, N-terminally acetylated; NH2, C-terminally amidated.


12

Supplementary Table 7. Concordant gene expression changes associated with the

effect of MAIT-203 treatment and the knockdown of APC in SW480 cells.

Common DEGs

SW480+MAIT-203 vs

SW480

SW480+shAPC vs

SW480

Symbol Gene_ID Up/Down

logCPM FDR

logCPM FDR

TM9SF1 10548 Up 0.13 3.71E08 0.73 0.002132

FAM157B 100132403 Up 0.57 0.008694 0.54 0.000522

FOSB 2354 Up 4.21 4.43E11 3.93 0.000409

SNAI1 6615 Up 3.33 0.00015 3.31 6.97E05

SGK1 6446 Up 3.03 0.006576 3.09 1.14E05

LOC100093631 100093631 Up 5.25 2.62E06 5.18 1.21E05

ZFP36 7538 Up 3.40 0.038826 3.38 0.004604

JUN 3725 Up 5.89 1.21E09 5.81 3.17E05

EGR1 1958 Up 6.68 1.96E06 6.73 3.23E11

PLK2 10769 Up 4.70 0.024302 4.79 2.06E06

CSRNP1 64651 Up 4.36 0.087952 4.44 5.60E06

ASB3 51130 Up 4.73 0.073484 4.81 6.53E06

HOXB2 3212 Up 4.90 0.036717 5.31 1.51E39

CYR61 3491 Up 5.63 0.00651 6.50 1.74E188

SNHG1 23642 Up 6.97 0.02045 7.16 3.84E20

PPP1R15B 84919 Up 5.78 0.038826 5.79 0.001993

PPP1R15A 23645 Up 6.27 0.016998 6.52 8.29E27

MYOF 26509 Up 7.70 0.021479 7.66 0.078771

TNFRSF10D 8793 Up 6.34 0.044123 6.64 4.58E41

IER2 9592 Up 6.62 0.053839 6.81 2.03E21

SPRY4 81848 Up 7.73 0.037896 7.83 2.67E12

HSP90AA1 3320 Up 12.03 0.034385 12.21 6.18E31

PVRIG2P 101752334 Down 1.65 2.66E33 1.82 3.37E05

TMEM238 388564 Down 1.91 5.48E05 1.89 3.02E07

PIK3R2 5296 Down 5.13 0.084158 5.28 0.000875

ZMAT1 84460 Down 2.69 0.047061 2.73 0.006319

HAUS4 54930 Down 4.65 0.002403 4.65 5.13E06

FAM89B 23625 Down 4.53 0.036717 4.36 1.12E14

MEGF6 1953 Down 5.38 0.073484 5.35 4.66E06

COL16A1 1307 Down 5.11 0.073484 4.95 1.85E17

OBSCN 84033 Down 5.80 0.054186 5.74 6.78E10

ST14 6768 Down 6.61 0.01828 6.20 2.02E100

TXNIP 10628 Down 7.04 0.045229 6.57 1.35E121


13

Supplementary Figures

Supplementary Figure 1. Overall view of the crystal structure of APC complexed

with Asef (PDB ID: 3NMZ). APC is drawn as solvent accessible surface colored by

gray and Asef is colored in purple. Both the N- and C-terminus of the Asef fragment

are labeled. Pocket surface of APC-ARM for Asef binding is colored in orange.


14

Supplementary Figure 2. Extensive residue-residue interactions on the interface of

the APC-ORGIN complex structure. The residues belonging to APC and ORIGIN are

labeled in green and blue, respectively. It was drawn using the LIGPLOT program. A

distance between donor and acceptor of less than 3.4 Å indicates a hydrogen bond,

and a 4.1 Å distance between two hydrophobic atoms indicates a hydrophobic

interaction.


15

Supplementary Figure 3. Crystal structures of APC/peptide complexes. (a) Crystal

structure of the APC/MAI-102 complex. APC is drawn as solvent accessible surface

colored by gray, and MAI-102 is depicted as sticks (carbon atoms: cyan). (b) Crystal


colored by gray, and MAI-107 is depicted as sticks (carbon atoms: violet). (c) Crystal


colored by gray, and MAI-108 is depicted as sticks (carbon atoms: pink). R549 in

APC is highlighted in all panels and colored in orange.


16

Supplementary Figure 4. Fo-Fc omit map for each MAI compound in complex

structures contoured at 3σ. (a) MAI-102 complex. (b) MAI-107 complex. (c) MAI-

108 complex. (d) MAI-150 complex. (e) MAI-203 complex.


17

Supplementary Figure 5. Conformational comparison of MAI-102, MAI-107, and

MAI-108 in the APC-ARM pocket. The red lines denote the distance between the

nitrogen atom of R549 in APC and the oxygen atoms of MAI-102, MAI-107, or MAI-

108.


18

Supplementary Figure 6. Structural comparison of MAI-102 (carbon atoms in cyan),

MAI-107 (carbon atoms in violet), MAI-108 (carbon atoms in pink), and MAI-150

(carbon atoms in yellow) in the APC-ARM pockets. Black arrow represents the shift

of C-terminus in MAI-150 compared to that in other compounds.


19


20

Supplementary Figure 7. In vitro binding of APC (303–739) mutants against MAI-

150 by Isothermal Titration Calorimetry (ITC) experiments. (a) APC-D539A. (b)

APC-K586A. (c) APC-N507W. (d) APC-K516A. (e) APC-W593A. (f) APC-R549A.

N indicates the number of sites per APC, K represents the binding constants between

APC and peptides, ∆H indicates heat change, ∆S indicates entropy change and N/A

indicates that the data could not be determined. Each ITC experiment was done once.


21

Supplementary Figure 8. In vitro binding of APC (303–739) against

peptidomimetics by ITC. (a) MAI-100. (b) MAI-201. (c) MAI-202. (d) MAI-204. N

indicates the number of sites per APC, K represents the binding constants between

APC and peptides, ∆H indicates heat change, ∆S indicates entropy change and N/A

indicates that the data could not be determined. Each ITC experiment was done once.


22

Supplementary Figure 9. Co-immunoprecipitation assays show the disruption of

APC-Asef interaction in the presence of MAI-203 and MAI-150 in the lysate of

HEK293T cells transfected with Flag-APC (303–876) and HA-Asef (170–632). (a)

Lysates from HEK293T cells were treated with DMSO, 0.1 µM, 0.5 µM, 2 µM, 5 µM

or 10 µM MAI-150 or MAI-203. (b) HEK293T cells were treated with DMSO, 50

µM, 100 µM or 200 µM MAI-150 or HEK293T cells lysates were treated with

DMSO or 10 µM MAI-150. Western blots were developed using an anti-HA or an

anti-Flag antibody, as shown. Full blots can be found in Supplementary Figure 29 and

Supplementary Figure 30.


23

Supplementary Figure 10. Internalization of FITC-labeled MAIT compounds into

cells. (a) HEK293T cells. (b) HCT116 cells. Dark blue curves for DMSO, pink curves

for TAT-FITC, light blue curves for MAIT-100-FITC, blackish green curves for

MAIT-150-FITC and orange curves for MAIT-203-FITC. Representative of two

independent experiments.


24

Supplementary Figure 11. Cell penetration by MAIT-150-FITC and MAIT-100-

FITC in HEK293T cells, SW480 cells, and HCT116 cells. Cellular penetration of

MAIT-100-FITC and MAIT-150-FITC was assessed by fluorescence microscopy.

Cells were incubated with DMSO, 25 µM MAIT-100-FITC, or 25 µM MAIT-150-

FITC, washed and imaged. Scale bars, 50 µm. Representative of two independent

experiments.


25

Supplementary Figure 12. Disruption of APC-Asef interaction by MAIT-150

determined using co-immunoprecipitation (co-IP) assays. HEK293T cells transfected

with Flag-APC (303–876) and HA-Asef (170–632) were treated with DMSO, 50 µM

MAI-150, 50 µM TAT, 25 µM or 50 µM MAIT-150 in the medium for 24 h. Western

blots were developed using an anti-HA or an anti-Flag antibody. Full blots can be

found in Supplementary Figure 31.


http://www.nature.com/nchembio/journal/v12/n12/full/nchembio.2209.html#supplementary-information

26

Supplementary Figure 13. Representative immunoblots for the CETSA assay

carried out in SW480 cells treated with 25 µM MAIT-150 (+) or DMSO (–). Western

blots were developed using an anti-APC antibody. Full blots can be found in

Supplementary Figure 32. Representative of three independent experiments.


27

Supplementary Figure 14. Specificity of MAIT-203 in the disruption of APC-Asef

interaction in HEK293T cells. HEK293T cells transfected with Flag-APC (303–876)

and Sam68-HA or Striatin(415–780)-HA were treated with DMSO, 10 µM MAIT-

100, or 10 µM MAIT-203 in the medium for 24 h. Western blots were developed

using an anti-HA or an anti-Flag antibody. Full blots can be found in Supplementary

Figure 33.


28

Supplementary Figure 15. Bar graphs for the inhibition of migration and invasion of

SW480 cells treated with MAITs. (a) Histogram of the migration of DMSO, MAIT-

100, or MAIT-203 treated SW480 cells assessed by xCELLigence RTCA-DP. Error

bars represent s.d., n 4. (b) Histogram of wound healing assays on DMSO, MAIT-

100, or MAIT-203 treated SW480 cells under phase contrast microscopy. Error bars

represent s.d. of triplicate experiments. (c) Histogram of transwell migration assays

on a chamber of DMSO, MAIT-100, or MAIT-203 treated SW480 cells. Error bars

represent s.d.. Experiment was repeated three times. (d) Histogram of the invasion of

SW480 cells measured by xCELLigence RTCA-DP. Error bars represent s.d., n 4.

Differences between individual groups were analyzed using t-tests (two-tailed and

unpaired). ns, nonsignificant, P values of <0.05 (*), <0.01 (**) or <0.001 (***) are

considered to be significant. All raw data for histograms were present in Fig. 5.


29

Supplementary Figure 16. Inhibition of the migration of SW480 cells and HCT116

cells treated with MAIT-203 or MAIT-150 by RTCA-DP. SW480 (a) and HCT116 (b)

cells were treated with 10 µM MAIT-203 or MAIT-100, 25 µM MAIT-203 or MAIT-

100. SW480 (c) and HCT116 (d) cells were treated with 25 µM MAIT-150 or MAIT-


30

100, 50 µM MAIT-150 or MAIT-100. The migration assay was conducted using

xCELLigence RTCA-DP, and the kinetic curves were recorded in real time. Error

bars represent s.d., n4. Differences between individual groups were analyzed using t-

tests (two-tailed and unpaired). ns, nonsignificant, P values of 0.05 (*), 0.01 (**) or

0.001 (***) are considered to be significant.


31

Supplementary Figure 17. Inhibition of the migration of SW480 cells treated with

MAIT-203 by wound-healing assay. Representative images of DMSO, MAIT-100(10

µM, 25 µM), or MAIT-203(10 µM, 25 µM) treated SW480 cells under phase contrast

microscopy. Scale bar represents 200 μm. Error bars represent s.d. of triplicate

experiments. Differences between individual groups were analyzed using t-tests (two-

tailed and unpaired). ns, nonsignificant, P values of 0.05 (*),0.01 (**) or 0.001

(***) are considered to be significant.


32

Supplementary Figure 18. Inhibition of the migration of HCT116 cells treated with

MAIT-203 by wound-healing assay. Representative images of DMSO, MAIT-100 (10

µM, 25 µM), or MAIT-203 (10 µM, 25 µM) treated HCT116 cells under phase

contrast microscopy. Scale bar represents 200 μm. Error bars represent s.d. of

triplicate experiments. Differences between individual groups were analyzed using t-

tests (two-tailed and unpaired). ns, nonsignificant, P values of 0.05 (*), 0.01 (**) or

0.001 (***) are considered to be significant.


33

Supplementary Figure 19. Inhibition of the migration of HCT116 cells treated with

MAIT-203 by transwell assay. Representative images of migration assays on a

chamber of DMSO, 25 µM MAIT-100, 10 µM MAIT-203, or 25 µM MAIT-203

treated HCT116 cells and folds of migrated HCT116 cells were calculated. Scale bar

represents 100 μm. Error bars represent s.d.. Experiment was repeated three times. ns,

nonsignificant. P values of 0.05 (*), 0.01 (**) or 0.001 (***) are considered to be

significant.


34

Supplementary Figure 20. Morphology of SW480 and HCT116 cells treated with

medium, DMSO, MAIT-100, MAIT-150, or MAIT-203 at the indicated

concentrations. (a) Microscopy images showing the morphology of SW480 cells

treated with medium, DMSO, 50 µM MAIT-100, 50 µM MAIT-150, or 25 µM

MAIT-203 for 0 h, 24 h, 48 h, and 72 h. (b) Microscopy images showing the

morphology of HCT116 cells treated with medium, DMSO, 50 µM MAIT-100, 50

µM MAIT-150, or 25 µM MAIT-203 for 0 h, 24 h, and 48 h. Imaged using a 10

objective. Scale bar, 50 µm. Representative of two independent experiments.


35

Supplementary Figure 21. MTS assays on cell proliferation after the treatment of

peptidomimetics in colorectal cancer cells. (a) SW480 cells (b) HCT116 cells. Cells

were treated with medium (blank), DMSO, 50 µM, and 100 µM MAIT-100, MAIT-

150 or MAIT-203. Cell proliferation was normalized to the DMSO treatment

group. n = 4 wells of samples for each condition. The experiment was repeated twice.


36

Supplementary Figure 22. Bar graph of relative GEF activity of Asef on CDC42

calculated based on figure 6b at 400s, 800, and 1200s. The labels 400S, 800S, and

1200S refer to the time points since the mixture of mant-GTP and CDC42 in the GEF

activity assay, and the y-axis bar values of relative activity represent the GEF activity

of Asef on CDC42 in Fig.6b as calculated in Materials and methods. Error bars

represent s.d. from three independent experiments. ns, nonsignificant. P values of

0.05 (*), 0.01 (**) or 0.001 (***) are considered to be significant.


37

Supplementary Figure 23. GEF activity of Asef toward Rac1. (a-b) Asef failed to

catalyze nucleotide exchange on Rac1 expressed by baculovirus system at the

indicated concentration by adding either Asef alone (a) or APC-Asef complex (b). (c)

Asef failed to catalyze nucleotide exchange on Rac1 expressed by bacterial system at

the indicated concentration. The fluorescence-based guanine nucleotide exchange

factor (GEF) activity assay was used to analyze the stimulation of Asef’s GEF activity

for CDC42 or Rac1. Relative fluorescence from one representative of two

independent experiments.


38

Supplementary Figure 24. Full Western blot images for Fig.4b.


39

Supplementary Figure 25. Full Western blot images for Fig.4d.


40

Supplementary Figure 26. Full Western blot images for Fig.6a.


41

Supplementary Figure 27. Western blots showing the knockdown efficiency of the

shRNA of APC in SW480 cells. Cells were infected with the vectors containing

oligonucleotides encoding shRNA-APC or vector (shRNA-NC). Lysates prepared

from infected cells were analyzed by immunoblot with antibodies against APC.

Antibodies against β-Actin were used as a control. Full blots can be found in

Supplementary Figure 34.


http://www.nature.com/nchembio/journal/v12/n12/full/nchembio.2209.html#supplementary-information

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44

Supplementary Figure 28. Purification and mass spectrometry analysis of peptides

and peptidomimetic. HPLC (Left) and MS profiles (Right) of MAI-100, MAI-102,

MAI-107, MAI-108, MAI-150, MAI-203, MAIT-100, MAIT-150, and MAIT-203

document compound purity (>95%) and mass identity, respectively.


45

Supplementary Figure 29. Full Western blot images for Supplementary Figure 9a.


46

Supplementary Figure 30. Full Western blot images for Supplementary Figure 9b.


47

Supplementary Figure 31. Full Western blot images for Supplementary Figure 12.


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