β-arrestin1 mediates metastatic growth of breast cancer cells by facilitating HIF-1-

dependent VEGF expression

Sudha K. Shenoy§1,2, Sang-oh Han1, Yu Lin Zhao3, Makoto R. Hara1, Timothy Oliver2, Yiting

Cao3 and Mark W. Dewhirst3

From the Departments of 1Medicine, 2Cell Biology, and 3Radiation Oncology, Duke University

Medical Center, Box 103204, Durham, North Carolina 27710 Phone: (919) 681-5061; Fax (919)

681-7851

§Corresponding author, Sudha K. Shenoy: sudha@receptor-biol.duke.edu

Supplementary Information:

Supplementary Table I

Table lists the details of p53, ER and PR expression in IDC and metastatic IDC samples

as provided by IMGENEX. The levels of β-arrestin1 was determined by immunostaining (+

equivalent to the levels in normal breast sample, the increased levels were assigned according to

the signal intensities quantified with MetaMorph (+: similar to normal breast, ++ up to 2-fold

increase, +++, up to 3-fold and ++++ > 3-fold)

SAMPLE # Sex/age Tissue Diagnosis Tumor size(cm)

ER PR p53 beta-arrestin

Imgenex:1 Female/28 Breast IDC 8 + - + ++Imgenex:4 Female/43 Breast IDC 4 + + - ++Imgenex:5 Female/39 Breast IDC 6.8 - - + ++Imgenex:6 Female/46 Breast IDC 5.5 + + - +++Imgenex:7 Female/47 Breast IDC 4.7 - - + ++Imgenex:8 Male/67 Breast IDC 6.5 + + + ++Imgenex:9 Female/33 Breast IDC 5.5 + + - +++Imgenex:10 Female/47 Breast IDC 2.5 + + - ++Imgenex:12 Female/46 Breast IDC 5.5 + - - ++++Imgenex:13 Female/39 Breast IDC 3 + ND - ++Imgenex:16 Female/58 Breast IDC 4.58 - - - +Imgenex:17 Female/37 Breast IDC 2.8 + + + ++Imgenex:18 Female/43 Breast IDC 4 - - + +++Imgenex:21 Female/36 Breast IDC 4.5 - - - +++Imgenex:22 Female/59 Breast IDC 4.5 + + - ++Imgenex:24 Female/54 Breast IDC 4 + + + +Imgenex:25 Female/47 Breast IDC 2.5 + + - ++Imgenex:26 Female/53 Breast IDC 3.5 - - + +Imgenex:27 Female/59 Breast IDC 4 + - - ++++Imgenex:29 Female/37 Breast IDC 5 - + + +++Imgenex:30 Female/46 Breast IDC 3 + - - +++Imgenex:31 Female/35 Breast IDC 5.5 + - - +++Imgenex:32 Female/47 Breast IDC 4.5 - - + ++++Imgenex:33 Female/54 Breast IDC 4.9 + + - +Imgenex:34 Female/47 Breast IDC 3 - - + +Imgenex:35 Female/41 Breast IDC 7 + + - +Imgenex:36 Female/38 Breast IDC 8 - - + ++Imgenex:37 Female/55 Breast IDC 3.5 - - + ++Imgenex:38 Female/65 Breast IDC 3 + - - +++Imgenex:39 Male/66 Breast IDC 5.5 - - - ++Imgenex:40 Female/44 Breast IDC 5.5 + - + +++Imgenex:41 Female/52 Lymph node m-IDC 5 - - + +++Imgenex:42 Female/32 Lymph node m-IDC 7 + + - ++Imgenex:43 Female/58 Lymph node m-IDC ND + + + +Imgenex:45 Female/58 Lymph node m-IDC 3.5 - - + +Imgenex:47 Female/45 Lymph node m-IDC 2.5 - - - ++Imgenex:48 Female/47 Lymph node m-IDC 4.5 + + - +++Imgenex:49 Female/47 Lymph node m-IDC 4.5 - - + +++

Supplementary Figure 1

Immunostaining of human breast tissue sections (Zymed breast tissue arrays) with different anti-

β-arr antibodies: Tissue sections were stained with either anti-βarr1 (A1CT), monoclonal anti-

βarr1 (BD Biosciences) or anti-βarr2 (A2CT) antibodies, followed by secondary antibody

conjugated to Alexa-Fluor 488 (Green channel). Shown are representative confocal images of

IDC samples. Also shown is a representative image where the same immunostaining procedure

was performed only with secondary antibody and no primary antibody was added. Nuclei

labeled with DRAQ5TM are shown in blue.