Additional file 1: Figure S1. of ADA3 regulates normal and tumor mammary epithelial cell proliferation through c-MYC
Creators: name: Griffin, Nicolas
name: Sharma, Gayatri
name: Xiangshan Zhao
name: Mirza, Sameer
name: Shashank Srivastava
name: Dave, Bhavana
name: Aleskandarany, Mohammed
name: Rakha, Emad
name: Shakur Mohibi
name: Band, Hamid
name: Band, Vimla
Publisher: name: Figshare
Year: 2016
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Computational Biology

description: High ADA3 expression in patient derived xenografts correlates with elevated Ki67. Are presentative of two ER+ /PR+ tumor grafts from two different breast cancer patients (HCI-003 and HCI-011). Patient-derived xenografts (as described in DeRose et al. [11]) were sectioned and stained with antibodies indicated (brown staining). Blue staining is hematoxilin. Figure S2. Knockdown of ADA3 causes a delay in cell cycle progression in immortal hMECs.76N-TERT cells expressing scrambled shRNAor Ada3 shRNAwere deprived of growth factors in DFCI-3 media for 72hrs, then released from synchrony by adding growth factor containing DFCI-1 medium. Cells were then collected at the indicated time points for various analyses. (A) FACS analysis after propidiumiodide staining. Cell cycle profile (G1/S/G2/M) at selected time points is shown (B) Lysates were immunoblotted with indicated antibodies. β-actin was used as a loading control. (C) Lysates from 76N-TERT transfected with ctrl. or ADA3 siRNA were immunoblotted with indicated antibodies. Figure S3. Exogenous overexpression of ADA3 in Human Mammary Epithelial Cells. Endogenous expression of ADA3 in 76N-TERT cells is only nuclear but after over expression of ADA3, expression is observed in both nucleus and cytoplasm. (A) 76N-TERT.V (i and ii) or 76N-TERT. ADA3 cells (iii and iv) stained for ADA3. (B) ADA3 protein levels after biochemical fractionation, PARP and α-tubulin are used as nuclear and cytoplasmic control, respectively. Figure S4. ADA3 over expression does not alter chromosomal stability. A) Representative karyotype of 76N-TERT vector cells and B) representative karyotype of 76N-TERT ADA3 cells. Figure S5. ADA3 overexpression does not induce estrogen independence for proliferation. Indicated cell lines overexpressing vector (V) or ADA3 were deprived of estrogen in phenol red-free alpha-MEM medium supplemented with 5% charcoal dextran stripped fetal calf serum for 72hrs and stimulated with 1nM of β-estradiol. Fresh medium was added on alternate days and total number of cells were determined using a hemocytometer and trypan blue exclusion method. Figure S6. ADA3 overexpression has no effect on invasion or migration of cells. ZR-75-1, a ER+ breast cancer cell line expressing vector (V) or ADA3 was examined for changes in invasive (A) or migratory (B) potential using Boyden chamber method. Table S1. Karyotypicanalysis of 76N-TERT vector or ADA3 overexpressing cell lines. Table S2. Survival analysis in the whole cohort. Table S3. Survival analysis in the ER+ cohort Only. (PDF 575 kb)