Supplemental Figure 1

1.2 1.2

BT474 MDA-MB-231

0 4

0.6

0.8

1

0 4

0.6

0.8

1

elat

ive

Abs

.

elat

ive

Abs

.

*

*

*

0

0.2

0.4

DMSO TCDD�10�nM

MCDF�5�uM

0

0.2

0.4

DMSO TCDD�10�nM

MCDF�5�uM

Re

Re

Supplemental Figure 1. Cell growth inhibition. Cells were treated with DMSO, TCDD or MCDF, and the effects on proliferation using the MTT assay were determined as described in the Materials and Methods Results are expressed as means � SE for 3 replicateMethods. Results are expressed as means � SE for 3 replicatedeterminations, and significant (p<0.05) inhibition is indicated (*).

Supplemental Figure 2

CTL TCDD (20 nM) MCDF (5 �M)

MCDF (5 �M)+ SOX4

TCDD (20 nM)+ SOX4

Supplemental Figure 2. MDA-MB-231 cell migration. MDA-MB-231 cells were treated with DMSO (CTL), 20 nM TCDD, or 5 �M MCDF alone or in combination with SOX4 overexpression, and after 36 hr cell migration was determined in a scratch assay as outlined in the Materials and MethodsMaterials and Methods.

Supplemental Figure 3

CTL TCDD (20 nM) MCDF (5 �M)

MCDF (5 �M)+ SOX4

TCDD (20 nM)+ SOX4

CTL+ SOX4

Supplemental Figure 3. MDA-MB-231 cell invasion. MDA-MB-231 cells were treated with DMSO (CTL), 20 nM TCDD, or 5 �M MCDF alone or in combination with SOX4 overexpression, and after 48 hr

SOX4

alone or in combination with SOX4 overexpression, and after 48 hr cell invasion was determined in a scratch assay as outlined in the Materials and Methods.

Supplemental Figure 4

Input IgG AHR

MDA-MB-231

Input IgG AHR

335-DRE1-4

MDA-MB-231

335-DRE5-6

335-DRE7

Cyp1A1 (-500)

AHR

Cyp1A1

�-actin

Supplemental Figure 4 CHIP assay for AHR-DRE interactions A

yp ( )

Supplemental Figure 4. CHIP assay for AHR DRE interactions. A. CHIP assay. MDA-MB-231 cells were treated with DMSO, 10 nMTCDD, or 5 �M MCDF for 1 hr and analyzed in a CHIP assay for AHR-DRE binding as described in the Materials and Methods. B. Induction of CYP1A1 protein. MDA-MB-231 cells were treated with 10 nM TCDD or 5 �M MCDF for 24 hr, and CPY1A1 protein was determined by western blot analysis as described in the Materials and Methods. �-Action served as a loading control.

Supplemental Figure 5

A B C DPercentage of Tumor to Total Lung

MCDF Treated

Mouse 1

NoMetastases Observed

Total Lung

546346 0

0%

Mouse 2

No

548244 2617

0.48%

Corn Oil (CTL)

Mouse 3

NoMetastases Observed

461728 0

0%

6 58%Mouse 1

Mouse 2

476532 31341

6.58%

8.44%Mouse 2

Mouse 3

453325 38257

12.34%

650620 80300

Supplemental Figure 5. Image analysis of mouse lung tissues. HE-stained slides were scanned to create a high-quality image (A) and all non-lung tissue was edited from the image (B). The image was converted into an 8-bit binary image (C) and the number of i l l l t d t t t l l N t ll l l ti dit dpixels was calculated to measure total lung area. Next, all normal lung tissue was edited

from the images so that only tumor metastases remained (D) to calculate tumor area and consecutively, the percentage of tumor per total lung volume. There is a clear difference in the pulmonary antimetastatic activity of MCDF in vitro when compared to mice gavaged with corn oil.