Supplementary Figure 1 A B 20 18 1 16 0.9 0.8 14 0.7 12 0.6 10 0.5 Number of Wells 8 0.4 0.3 6 0.2 4 0.1 Relative mRNA level for cells treated with different siRNAs 2 0 2 N B 1 5 1 2 S E R R H N C 0 R T P F D T A F P T G C C R 0.7 0.8 0.9 1 1.1 1.2 1.3 Control P F A G ene targeted and probed Normalized Sheet Migration

C

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

Normalized Sheet Migration Duplicate 2 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 Normalized Sheet Migration Duplicate 1 Supplementary Figure 2

B A 7 6 Domains 6 3 Functions 4 5

4 9 3 6 16 3 16 10 5 10

Fold Enrichment 2 C ell C ycle Focal Adhesion 1 FGFR Signaling R egulation 0 c

R hoA/Back/Contraction c J J n K S P O A i M A K A T T A T F H T

Vesicular Transport c 2 u _ P R L R A t P Other G S P Supplementary Figure 3

A B 0.6

d F GFR+(withFGFR+) r F GFR+(withFGFR+) a 0.55 F GFR-(withFGFR-) w Stained

o F GFR-(withFGFR-) 0.55 Unstained

T 0.5 F GFR-(withFGFR+)

s F GFR+(withFGFR-) t F GFR+(withFGFR-) 0.5 n 0.45

e F GFR-(withFGFR+) F GFR-(withFGFR+) m 0.45

e 0.4 F GFR+(withFGFR-)

v F GFR+(withFGFR-) o F GFR-(withFGFR+) 0.4

M 0.35

n

o 0.35 i 0.3 t c a r 0.25 0.3 F 0.25 0.2 ______FGFR1 -100 100 300 500 700 Directed Movements (at boundary) + + + + + + Distance from Edge(um) D +FGF C d

r 0.48 Unmixed FGFR+ a 0.4 2.0-fold Unmixed CDH- 1.7-fold w - R o Unmixed FGFR-

F 0.43

0.35 T

G Mixed FGFR+ F s / t

+ 0.3

n Mixed FGFR- R 0.38 F e

G 0.25 Mixed CDH- F m

o e Mixed FGFR- i

t 0.2 0.33 v a o R 0.15 t t M r r e e

e e e e

g g 0.28 e e e e n d d h h n n E E o o o S S i i i t P P c 0.23

FGFR+ Unstained FGFR+ Stained a r F 0.18 0 100 200 300 400 500 600 700 Distance from Edge(um) Supplementary Figure 4

12 Confluent Cells Sparse Cells ) r 11.5 u o h / m u

( 11

y t i c o l

e 10.5 V

l l e C 10

9.5 0.0001 0.01 1 100 10000 [FGF] ng/ml Supplementary Figure 5

C A Cell Density Module Single Cell Motility Module

PFN1 CCND1 RAC2 SH2B LOC284441 KIF11 ACTR2 MAP4K4 PTK9 CDK4 WAS RACGAP1 ARPC2 CD2AP CDC42 ROCK1 CRK RHOF CDKN1A PLCXD1 ACTN1 ARHGDIG

A c t i n TRIB1 P e r s i t P a x i l n M i c T u b p - M y o I OSBP2 A d h s S i z e C e l E o n g

A d h s R a t e OTUB1

C e l o n t a c MAGI1 ACTR3 CACNB4 PPP4R2 Cell-Cell Coordination Module RHOA B CAPN1 STK40 LOC201164 TAOK2 VCL AKT3 CTNNA1 ARHGDIA RAB27B RPS6KA1 DLG1 PTPRJ MX2 ANKRD13D MYO3A SCML4 PTPN14 RND2 ARHGAP29 MAGI3 PPP1CB PLA2G6 ARHGEF7 LOC124685 STAM2 UBE2R2 TRDN STX16 PXN PI4K2B PRKD2 DEPDC1B STX17

IMAA A c t i n P e r s i t P a x i l n M i c T u b RPS6KB1 p - M y o I A d h s S i z e C e l E o n g KPNA2 A d h s R a t e

GOPC C e l o n t a c PPP1R9A RAB1A PAK2 PPP1CC KIF18A ANXA10 UBA6 ARL17P1 SH3YL1 A c t i n P e r s i t P a x i l n M i c T u b p - M y o I A d h s S i z e C e l E o n g A d h s R a t e C e l o n t a c Supplementary Figure 6

More Elongated 0.92

0.9

0.88

0.86

0.84 Cell Eccentricity

0.82

0.8

More Rounded 0.78 -4 -2 0 2 4 6 8 10 Normalized Directional Correlation Supplementary Table 1 A

KEGG FUNCTION SMART Domain Leukocyte Focal Adheren Transendothelial Tight Actin Adhesion Junction Migration Junctions RHO PP2Ac GuKc RAS S_TK PTP FGFR1 RAP1A FGFR1 RAP1A RHOA RAB27B PPP1CC MAGI3 RAB27B RPS6KA6 PTP4A1 WAS PTEN RHOA PXN MAGI3 RHOA PPP1CA CACNB4 RAP1A TRIB1 PTPRB PTK2 AKT3 PTPRB RHOA PTEN RHOF PPP1CB DLG1 HRAS WEE1 PTPRJ Actin PTK2 PTPRJ PTPN11 AKT3 CDC42 MAGI1 AKT3 PTPN11 ARPC2 RAC2 WAS CDC42 CDC42 RAC2 MAP4K4 PTPN14 RAC2 HRAS CDC42 PTK2 ACTN1 RND2 RPS6KA1 PTEN HRAS CRK ACTN1 ACTN1 CTNNA1 TAOK2 CRK PPP1CC CTNNA1 CTNNA1 HRAS RPS6KB1 PPP1CC PXN RAC2 RAC2 MAGI1 PAK2 PXN RHOA VCL ROCK1 CDK4 PRKD2 RHOA CAPN1 VCL MARK2 PAK2 PAK2 MYO3A PPP1CA PPP1CA STK40 CDC42 CDC42 TRIO ACTN1 ACTN1 ROCK1 PFN1