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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 1 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] 1 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. 2 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) 3 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 (n3; * 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). 4 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. 5