Supplemental Figure 1. Vav1 is expressed in a subset of tumor cell lines. Lysates for the

indicated pancreatic cancer or breast cancer (CA1D) cell lines were immunoblotted to detect

Vav1 and actin as a loading control.

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Supplemental Figure 2. Azathioprine does not inhibit migration by pancreatic tumor cells.

Vav1-expressing (DanG, Panc04.03, CFPAC, A) or non-expressing (BxPC3, PANC1, B) cells

were grown to confluence, pre-treated with azathioprine for 1.5 days, and assayed for cell

migration using a wound-healing assay. Representative photomicrographs are shown of

migratory endpoints (t=24 hours, or t=7 hours for Panc04.03 cells), with the white line indicating

the starting point. The distance migrated was quantified and is graphed as the mean +/- SEM of

at least 3 independent experiments. * p<0.05.

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Supplemental Figure 3. Vav1 is expressed in tumors from a genetic model of pancreatic

cancer. (A) Azathioprine inhibits invasive migration induced by mouse Vav1. PANC1 cells

were transfected with empty vector or mouse Vav1, treated with vehicle or azathioprine for 2

days, and seeded in a transwell migration assay. The percent of cells invaded across the filter was

scored. Mouse Vav1 promotes invasive migration, and is sensitive to azathioprine’s anti-invasive

effects. Graphed data represent the mean +/- SEM of 4 independent experiments. * p≤0.05, ns:

no statistically significant difference. (B) Primary tumors from the p48Cre/+; KRasG12D; p53Flox/+

mice were solubilized and immunoblotted for Vav1 and actin as a loading control. Panc1 cells

were used as a negative control. Positive controls were DanG cells, and Panc1 cells transfected

with Vav1. Vav1 is present in the majority of mouse pancreatic tumors.

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Supplemental Figure 4

A. Flank xenograft model

Cell line n Treatment Primary tumor sizeDanG(Vav1 positive)

17 0 mg/kg 766 +/- 74 mm3

17 5 mg/kg 672 +/- 81 mm3

BxPC3(Vav1 negative)

6 0 mg/kg 915 +/- 200 mm3

6 5 mg/kg 962 +/- 276 mm3

B. Orthotopic xenograft model

Cell line n Treatment Primary tumor sizeDanG(Vav1 positive)

10 0 mg/kg 810 +/- 122 mm3

9 5 mg/kg 611 +/- 88 mm3

L3.6(Vav1 negative)

8 0 mg/kg 1.00 +/- 0.08 g8 5 mg/kg 0.97 +/- 0.11 g

C. Genetic mouse model: p48Cre/+; KRasG12D/+; p53Flox/+

n Primary tumor size (g) Survival (weeks)0 mg/kg 22 1.20 +/- 0.15 22.4 +/- 0.75 mg/kg 19 1.55 +/- 0.21 22.9 +/- 1.010 mg/kg 23 1.61 +/- 0.21 24.5 +/- 1.1

D. Genetic mouse model: PDX-Cre; KRasG12D; p53Flox/Flox

n Primary tumor size (g) Survival (weeks)0 mg/kg 12 0.9 +/- 0.09 8.6 +/- 0.45 mg/kg 12 0.8 +/- 0.04 8.0 +/- 0.315 mg/kg 12 0.9 +/- 0.13 8.3 +/- 0.2

E. Genetic mouse model: PDX-Cre; KRasG12D; p53Flox/R172H

n Primary tumor size (g) Survival (weeks)0 mg/kg 4 0.5 +/- 0.08 7.6 +/- 0.55 mg/kg 4 0.4 +/- 0.10 9.0 +/- 0.7

All data represent the mean +/- S.E.M.

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Supplemental Figure 4. Azathioprine treatment does not impact tumor size in mouse

models of pancreatic cancer. (A) DanG or BxPC3 tumor cells were implanted subcutaneously

into the flank of athymic nude mice, and mice were treated three times weekly with vehicle or

azathioprine for 3 weeks (DanG) or 5 weeks (BxPC3), and the volume of the primary tumor was

monitored. (B) DanG or L3.6 tumor cells were injected into the head of the pancreas of athymic

nude mice in an orthotopic tumor model, and mice were treated three times weekly with vehicle

or azathioprine for 3 weeks (DanG) or 5 weeks (L3.6). Upon necropsy, primary tumor size was

measured by volume (DanG) or mass (L3.6). (C-E) Multiple genetic mouse models for

pancreatic cancer, treated with vehicle or azathioprine three times weekly beginning at 5 weeks

of age until sacrifice. (C) While metastasis was inhibited in the p48Cre/+; KRasG12D; p53Flox/+ mice,

azathioprine did not impact primary tumor size. (D-E) The more aggressive mouse models PDX-

Cre; KRasG12D; p53Flox/Flox or PDX-Cre; KRasG12D; p53Flox/R172H had a mean survival time of only

approximately 8 weeks, and did not develop metastases. Azathioprine had no significant effect

on primary tumor size or survival time. All data represent the mean +/- SEM of the indicated

number of mice (n).

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Supplemental Figure 5. Azathioprine reduces cell propagation in a Vav1-independent

manner. (A) Cultured pancreatic cancer cells were incubated with azathioprine for 4 days, then

counted on a hemocytometer in the presence of trypan blue. Azathioprine treatment reduced the

total viable cell number after four days (normalized to vehicle-treated cells), in both Vav1-

positive and Vav1-negative cells. (B) The percent of trypan blue-positive dead cells was

determined. Azathioprine did not induce significant cell death after four days in either the Vav1-

positive or Vav1-negative cells. (C) Cells were treated as described in A, but after 4 days were

analyzed by MTS assay. The absorbance at 490 was measured in duplicate samples and

normalized to vehicle-treated cells for each cell type. All graphed data represent the mean +/-

SEM of 3 independent experiments. * p<0.05, ** p<0.01.

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