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SUPPLEMENTARY FIGURES, TABLES AND REFERENCES
The nuclear coactivator Amplified In Breast Cancer 1 maintains tumor
initiating cells during development of
Ductal Carcinoma In Situ
V Ory1, E Tassi1, LR Cavalli1, GM Sharif1, F Saenz1, T Baker1, MO Schmidt1, SC Mueller1,
PA Furth1, A Wellstein1 and AT Riegel1
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Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University,
Washington, DC, 20057
Correspondence: Dr AT Riegel, Department of Oncology, Lombardi Comprehensive Cancer Center,
Georgetown University, 3970 Reservoir Road NW, Washington, DC, 20057, USA. E-mail:
[email protected]
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Supplemental Materials and Methods
Boyden Chamber Migration Assay. MCFDCIS infected with lentivirus shCTRL or shAIB1
were seeded in quadriplate at a density of (12 × 104 cells/well) in the top chamber of a CIMplate 16 (Roche Diagnostics, Burgess Hill, West Sussex, UK) with Boyden-like chambers
coupled with the RTCA xCELLigence system following the manufacturer’s instructions, and
incubated at 37°C overnight. Data analyses were carried out with the RTCA Software 1.2.1 as
described elsewhere1,2.
Electric Cell-substrate Impedance Sensing system-based invasion assay. Human
umbilical vein endothelial cells (HUVEC) were plated (4 × 104 cells/well) in wells of an electric
cell-substrate impedance sensing systems (ECIS) array (E-19; Applied BioPhysics, Troy, NY)
precoated with a solution of 200 µg/mL gelatin in 0.15 mol/L NaCl. The HUVECs were
challenged with monodisperse cell suspensions of shCTRL and shAIB1 MCFDCIS cells and
MDA-MB-231 cells (2 × 104 cells/well) in fresh HUVEC medium. After adding breast cancer
cells, invasion into the HUVEC monolayer was followed by impedance measurements and
analyzed using ECIS software (Applied BioPhysics, Troy, NY) as described2,3. Triplicate wells
were used for each sample.
Supplemental References
1.
Rosenfield SM, Bowden ET, Cohen-Missner S, Gibby KA, Ory V, Henke RT et al.
Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and
Mammary Epithelial Cells. PLoS ONE 2012; 7:e47876.
2.
Keese CR, Bhawe K, Wegener J, Giaever I. Real-time impedance assay to follow the
invasive activities of metastatic cells in culture. BioTechniques 2002; 33:842–4, 846, 848–
50.
3.
Al-Otaiby M, Tassi E, Schmidt MO, Chien CD, Baker T, Salas AG et al. Role of the
nuclear receptor coactivator AIB1/SRC-3 in angiogenesis and wound healing. The
American Journal of Pathology 2012; 180:1474–1484.
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Supplemental Figures
Fig. S1. (A) Representative images of IHC staining for AIB1 in ER+ human comedo DCIS not
associated with invasive lesions (a-d). Scale bar = 0.2 mm. (B) Immunofluorescence images of DCIS
lesions stained with AIB1 antibody (5E11, Cell Signaling Technology, 1:75)(red signals), and Dapi (blue
signals) in MCFDCIS xenograft tissue sections, and analyzed with an Olympus FV300 Confocal
microscope equipped with Fluoview 300 Software. Scale bar = 20 μm. (C) Representative metaphase
spread of MCFDCIS cells hybridized with bacterial artificial chromosome clones (RP11-62N23)
containing sequences of the HER2/NEU gene. Note two copies of the HER2/NEU gene (green signals)
and centromeric control probe for chromosome 17 (RP11-299G20)(red signals) both in the metaphase
and interphase nuclei. Scale bar = 20 μm. (D) staining with PR antibody (C-19, Santa Cruz
Biotechnology, 1:100) in DCIS lesions in MCFDCIS xenograft tumors. Insets outline a zoom-in of the
framed areas. Scale bar = 0.1 mm.
Fig. S2. (A) Western blot analysis using antibodies against ER (Ab-15, Thermo Scientific, 1:200) and
β-actin (C4 Millipore, 1:3000) in MCFDCIS and MCF-7 cells. Actin was used as loading control. (B)
Representative picture of IHC staining with ER antibody (Ab-15, Thermo Scientific, 1:200) (left),
percentage of ER-positive nuclei measured per field in at least five fields non-overlapping using
Photoshop CS3 software (right). (C) Representative H&E stained images and (D) growth curve analysis
of MCFDCIS xenograft tumors in control and ovariectomized athymic nude mice. Tumor volume was
measured twice weekly with a caliper. Mice were euthanized by CO2 inhalation when the tumor size
reached a size greater than 500 mm3 or was necrotic. Mean  SEM (control group, n=16;
ovariectomized group, n=24; no significant difference vs. control). (E) Schematic representation of the
treatment of the MCFDCIS xenograft mouse model with fulvestrant. Mice were injected with control
vehicle or fulvestrant (200 mg/kg) twice a week after the establishment of DCIS lesions (14 days after
injection). Mice were euthanized when the tumor size reached greater than 500 mm3 or were necrotic.
(F) Representative cross-sections of H&E staining and (G) growth curve analysis of MCFDCIS
xenograft mice treated or not with fulvestrant. Mean  SEM (control group, n=10; fulvestrant-treated
group, n=9; no significant difference vs. control). DCIS lesions are highlighted with dotted line and in
H&E-stained slides. Scale bar = 0.2 mm.
Fig. S3. (A) Representative immunofluorescence pictures of MCFDCIS cell acini infected with lentiviral
shRNA control or shRNA AIB1 at day 7 using cleaved-caspase-3 antibody (Asp175, Cell Signaling
Technology, 1:100)(CC3, green signals) and Dapi (blue signals), and analyzed by confocal microscopy.
Scale bar = 20 μm. (B) Western blot analysis using AIB1, p21 (DCS60, Cell Signaling, 1:1000) and BIM
EL (Cell Signaling Technology, 1:1000) antibodies in protein lysates from MCFDCIS depleted or not of
AIB1. The levels of expression relative to -actin are indicated under each blot.
Fig. S4. (A) qRT-PCR analysis of human AIB1 (hAIB1) and -actin mRNA expression. (B)
Representative western blot (left) and quantification of AIB1 protein expression (right) in tumors from
control (shCTRL) and AIB1 depleted cells (shAIB1). AIB1 protein levels normalized against -actin are
indicated under each western blot. (C) Measurement of the DCIS lesion number per field in
representative shCTRL or shAIB1 xenografted tissue sections using Image J software. (D) Percentage
of p21-positive nuclei in shCTRL and shAIB1 tumors stained with an anti-p21 antibody (DCS60, Cell
Signaling, 1:1000) and measured in at least five non-overlapping fields using Photoshop CS3 software.
Mean  SEM (shCTRL, n=15; shAIB1, n=11; * p<0.01 vs. control; **, p<0.001 vs. control; *** p<0.0001
vs. control).
Fig. S5. (A) Representative phase-contrast (a-d) and tRFP images (e-h) of MCFDCIS cells infected
with tet-inducible shCTRL (a-b, e-f) and shAIB1 (c-d, g-h) lentiviral constructs. Scale bar = 0.2 mm. (B)
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Images of control (upper) and doxycycline-treated mice (bottom) using a CRi Maestro Imaging System.
Tumors are indicated by arrow heads. (C) qRT-PCR analysis of AIB1 mRNA and (D) western blot
analysis of AIB1 protein levels in MCFDCIS cells control (shCTRL –Dox, shCTRL +Dox, shAIB1 -Dox)
and depleted of AIB1 (shAIB1 +Dox). AIB1 protein levels normalized against -actin are indicated.
Mean  SEM (n=3; **, p<0.001 vs. control).
Fig. S6. (A) Representative phase-contrast pictures (left) and quantification of the spheres formed by
MCF-10A infected with lentivirus AIB1 or vector control in 50 non-overlapping fields using Photoshop
CS3 software (right). MCF-10A acini are indicated by arrow heads. Scale bar = 0.2 mm. Mean  SEM
(EV, n=50; AIB1, n=50; *, p<0.01 vs. control). (B) qRT-PCR analysis of AIB1, CD24, CD44 and CD49f
in MCF-10A cells overexpressing or not AIB1. Mean  SEM (EV, n=3; AIB1, n=3; *, p<0.01 vs. control,
**, p<0.001 vs. control). (C) Representative images of IHC staining for CD44 (a, b), CK18 (c, d), and
p63 (e, f) in tissue section of shAIB1 xenografted tumor tissues treated with doxycycline (b, d, f) or not
(a, c, e). Insets outline a zoom-in of the framed areas. Scale bar = 0.1 mm. (D) CK18 levels in DCIS
lesions constitutively depleted or not of AIB1. MetaMorph software was used for computerized
quantification of CK18-immunostained DCIS lesions. Percentage of p63-positive nuclei in shCTRL and
shAIB1 tumors (E) in the constitutive and (F) the conditional mouse model evaluated by measuring the
percentage of positive cells per field in at least five non-overlapping fields. Mean  SEM (shCTRL,
n=15; shAIB1, n=11; shAIB1-Dox, n=4, shAIB1+Dox, n=8; *, p<0.01 vs. control, ***, p<0.0001 vs.
control).
Fig. S7. (A) Representative western blot analysis for HER2 (06-562, Millipore, 1:700), HER3 (2F12,
Millipore, 1:200) and EGFR expression in protein lysates from MCFDCIS and MCF-7 cells in vitro. (B)
Analysis of HER2 protein expression by western blot in shCTRL and shAIB1 MCFDCIS cells. Protein
expressions are normalized to -actin. HER2 protein levels indicated under the western blot were
quantified using ImageJ 1.33u software and normalized to β-actin. (C) Quantification of HER2 mRNA
and (D) HER3 mRNA levels using qRT-PCR in MCFDCIS cells depleted (n=11) or not (n=15) of AIB1 in
the constitutive AIB1 knockdown MCFDCIS xenograft mouse model. (E) Western blot analysis and
quantification of HER2 and (F) HER3 expression in MCFDCIS shCTRL (n=10) and shAIB1 (n=8)
tumors using antibodies against HER2, HER3 and β-actin. (G) Representative western blot of EGFR
protein expression in tumors from control and AIB1 depleted cells. (H) qRT-PCR analysis of HER2
mRNA (left) and protein by western blot (right) in MCF-10A cells overexpressing or not AIB1
(EV=empty vector). (I) Real time PCR analysis of HER2 and AIB1 mRNA expression in MCFDCIS cells
stably infected with a lentivirus containing human HER2 (LVP504, GenTarget Inc) or the control vector
(EV), and co-infected with shRNA control or shRNA AIB1 lentiviral contructs. Mean  SEM (n3;
* p<0.01 vs. control; **, p<0.001 vs. control; *** p<0.0001 vs. control).
Fig. S8. (A) Boyden chamber assay showing migration of MCFDCIS infected with shRNA control (n=4)
or shAIB1 (n=4) quantified by electric cell substrate impedance sensing (ECIS). (B) ECIS-based
invasion assay through HUVEC cells by MCFDCIS cells deficient or not in AIB1 compare to the highly
invasive MDA-MB-231 cells (MDA-231) measured by the ECIS system. (C) Analysis of the
micrometastases in the lung, liver, brain and bone in control mice (n=3) or in MCFDCIS xenograft mice
6 weeks after injection of MCFDCIS cells (n=3) by qRT-PCR using primers specific to human -2microglobulin (2m) and mouse -actin. Ct values for human 2m gene are normalized to the mouse actin gene Ct values. Note that the raw human 2m Ct values are  30 and thus within background
reading. Mean  SEM (no significant difference vs. control).
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Supplemental Tables
Table S1. The table lists genes with significant changes in gene expression evaluated using microarray
analysis in MCFDCIS cells infected with control or AIB1 shRNA. Total RNA was harvested from
MCFDCIS cells transduced with lentiviral scrambled shRNA or shRNA directed against AIB1.
Microarray assays were performed in UCLA Neurosciences Genomics Core using Illumina Beadchip.
Microarray data were analyzed with R software. Microarray normalization by variance-stabilizing
transformation (VST) and background correction was carried out with the BioConductor package “lumi”.
The BioConductor package “limma” was used to complete differential gene expression analysis of the
normalized microarray data. Two independent experiments were run for each study group. Log2 fold
changes were calculated and a table ranking the top genes with an adjusted P value of p< 0.01 was
generated. The data represent fold differences genes that were significantly upregulated or
downregulated between the shAIB1 treated group and controls. Positive values indicate up-regulation
of individual genes; negative values indicate down-regulation.
Table S2. Primers used for qRT-PCR Analysis.
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