Supplementary Figure 1. Abercrombie's Confronted Explants Assay. The cell movement from the secondary explant is measured at four points 24h after contact with the primary explant. Three points measure the distance moved into free space while the fourth measures the movement of cells confronted by the primary explant. Invasion index = D/[(A+B+C)/3]. Complete loss of heterotypic CIL would give a value of 1.0 (never observed).

Supplementary Figure 2. RT-PCR of endogenous Rac1 and L61Rac1 transcripts in empty vector and L61rac1 transformed cell lines. A) L61rac1 (1), B) L61rac1 (2), C) empty vector line. GADPH mRNA levels are used as a loading control.

A B C

576 bp

574 bp

487 bp

GAPDH(26 cycles)

GAPDH(24 cycles)

Endogenous Rac1(34 cycles)

Endogenous Rac1(32 cycles)

L61Rac1(36 cycles)

L61Rac1(34 cycles)

Supplementary Figure 3. RT-PCR of endogenous Rac1 and N17Rac1 transcripts in N17Rac1 and empty vector (VV4) transformed NIH3T3 cell lines. GAPDH mRNA levels were used as a loading control. A) N17Rac1 (1), B) N17Rac1 (2), N17Rac (3), D) Empty vector line.

A B C D

576 bp

574 bp

487 bp

GAPDH (28 cycles)

GAPDH (25 cycles)

Rac1 Endogenous (38 cycles)

Rac1 Endogenous (36 cycles)

N17Rac1 (36 cycles)

N17Rac1 (34 cycles)

Supplementary Figure 4. RT-PCR of Tiam1 transcript levels in three NIH3T3 cell lines transformed with antisense Tiam1 compared with line transformed with the empty vector. A) Antisense Tiam1 (1), B) Antisense Tiam1 (2), C) Antisense Tiam1 (3), D) Empty vector line (pcDNA3.1). GAPDH transcript levels were used as a loading control.

576 bp

534 bp

A B C D

GAPDH (22 cycles)

GAPDH (20 cycles)

Endogenous Tiam1 (38 cycles)

Endogenous Tiam1 (36 cycles)

Supplementary Figure 5a. RT-PCR of endogenous Cdc42 and V12Cdc42 transcripts in V12Cdc42 and empty vector transformed NIH3T3 cell lines. GADPH mRNA levels were used as a loading control. A) V12Cdc42 (1), B) V12Cdc42 (2), C) empty vector line.

576 bp

636 bp

740 bp

A B C

GAPDH (26 cycles)

GAPDH (24 cycles)

Endogenous Cdc42 (35 cycles)

Endogenous Cdc42 (33 cycles)

V12Cdc42 (35 cycles)

V12Cdc42 (33 cycles)

Supplementary Figure 5b. RT-PCR of endogenous Cdc42 and N17Cdc42 transcripts in N17Cdc42 and empty vector transformed NIH3T3 cell lines. GADPH mRNA levels were used as a loading control. A) N17Cdc42 (1), B) N17Cdc42 (2), C) N17Cdc42 (3), D) empty vector line.

576 bp

636 bp

740 bp

A B C D

GAPDH (25 cycles)

GAPDH (23 cycles)

Cdc42 (32 cycles)

Cdc42 (30 cycles)

N17Cdc42 (34 cycles)

N17Cdc42 (32 cycles)

Supplementary Figure 6. RT-PCR of endogenous RhoA and V14RhoA transcript levels in three V14rhoA transformed cell lines and a line transformed with an empty vector (VV4). A)V14RhoA (1), B) V14RhoA (2), C) V14RhoA (3), D) Empty vector line.

576 bp

A B C D

GAPDH (24 cycles)

GAPDH (22 cycles)

RhoA Endogenous (32 cycles)

RhoA Endogenous (30 cycles)

V14 RhoA (32 cycles)

V14 RhoA (30 cycles)

734 bp

642 bp

Supplementary Figure 7. Changes in the levels of active RhoA in V14RhoA or antisense Tiaqm1 transfected NIH3T3 cells and in N17Rac1 cells and N17Rac1 cells transfected with p190RhoA or dominant active mDia. RhoA activity was assessed by the RhoA G-LISA Activation Assay. Absorbance at the control (c) was expressed as 1 arbitrary unit. Data was expressed as the means of three experiments with error bars showing S.E.M. P was < 0.01 compared with controls in all four experiments.A) Comparative RhoA-GTP levels in NIH3T3 cells transfected with an empty vector (VV4) or N17Rac1.B) Comparative RhoA-GTP levels in NIH3T3 cells transfected with an empty vector (VV4) or V14RhoA.C) Comparative RhoA-GTP levels in NIH3T3 cells transfected with an empty vector (pcDNA3.1) or antisense Tiam1.D) Comparative RhoA-GTP levels in N17Rac1 NIH3T3 cells transfected with a p190RhoA construct.E) Comparative RhoA-GTP levels in N17Rac1 NIH3T3 cells transfected with dominant-active mDia.

0.0

0.5

1.0

Abi

trar

y U

nit

N17Rac1(c)

p190RhoAN17Rac1

0.0

0.5

1.0

Abi

trar

y U

nit

N17Rac1(c)

N17Rac1d.a. mDia

Abi

trar

y U

nit

0

1

2

VV4 (c)

V14RhoA

B

0

1

2

Abi

trar

y U

nit

pcDNA3.1(c)

antisenseTiam1

CA

0

1

2

VV4(c)

N17Rac1

Abi

trar

y U

nit

D E

576 bp

350 bp

322 bp

A B C

GAPDH (18 cycles)

GAPDH (16 cycles)

Endogenous mDia (38 cycles)

Endogenous mDia (36 cycles)

DA mDia (32 cycles)

DA mDia (30 cycles)

Supplementary Figure 8a. RT-PCR of endogenous and dominant active mDia and compared with the parental line. GADPH transcript levels were used as a loading control. A) N17Rac1Rac1/DAmDia(1), B) N17Rac1/DAmDia(2), C) N17Rac1 control.

GAPDH (23 cycles)

GAPDH (21 cycles)

Endogenous mDia (39 cycles)

Endogenous mDia (37 cycles)

DA mDia (39 cycles)

DA mDia (37 cycles)

576 bp

350 bp

322 bp

A B C

Supplementary Figure 8b. RT-PCR of endogenous and dominant active mDia transcript levels in two NIH3T3 cell lines transformed with dominant active mDia and one line transformed with empty vector. GAPDH transcript levels were used as a loading control. A) DA mDia (1), B) DA mDia (2), C) NIH3T3/empty vector.