Supplementary material and methods Cell proliferation assay The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTT) assay (Promega) was used to assess cell proliferation, as described in our previous paper [1]. Western blot analysis Cells were serum starved for 24 hours and then treated with either vehicle control (DMSO) or various concentrations of LBH589. Cell lysates were run on a 12% acrylamide gel, transferred to a nitrocellulose membrane, and probed with antibodies. NF-κB1, pNF-κB1, RelA, p-RelA, and IκBα antibodies were purchased from Santa Cruz Biotechnology. -actin, cleaved-PARP, cFLIP, p21WAF1/CIP1, Bax, Bcl-xL, AKT, ERα and Lamin A/C antibodies were purchased from Cell Signaling Technology. Bands were visualized by chemiluminescence using HRP-conjugated secondary antibodies. Cell cycle analysis Cell lines were treated with LBH589 (20 nM) or vehicle control (DMSO) for the stated time periods. To include detached and floating apoptotic bodies, culture and wash media were all saved and included in the analysis. Cells were washed with PBS and detached using 0.25% trypsin-EDTA. Detached cells, culture medium, and the PBS wash were combined and spun for 4 minutes at 200 g. To fix the cells, 1 ml of -20°C 70% ethanol in H2O was added, drop-wise, while gently vortexing; cells were stored overnight at 4°C. On the day of staining, cells were washed with 1% BSA in PBS and then treated for 30 minutes with 250 μl of staining solution containing 50 μg/ml propidium iodide, 20 μg/ml RNase A, and 1% BSA in PBS. Cells were analyzed by flow cytometry using a FACScan (Becton Dickinson) flow cytometer equipped with a blue laser (488 nm) for the excitation source. Fluorescence emission was detected using 585/42 nm band pass filters. FACS data were analyzed using the cell cycle calculator in the ModFit LT 3.0 software package (Verity Software House). Animal studies Tumor specimens were stained using cleaved PARP antibody (Asp214, Cell Signaling Technology) for apoptosis and Ki-67 antibody (M7240, Dakocytomation) for cell proliferation. Antigen retrieval was done for 30 minutes in citrate buffer or EDTA buffer at 98 oC, respectively. Immunohistochemically stained slides were quantified by taking four random fields, counting stained and unstained cells, and dividing each by the total number of cells counted to generate the percentage of positive cells. To measure NF-B1 gene expression, total RNA was extracted from tumor specimens with TRIzol reagent (Invitrogen), and then cDNA synthesized from isolated RNA and analyzed by realtime PCR. Real-time PCR analysis For cell culture studies, cells were plated in 60 mm dishes at a density of 8x105 cells/dish and treated with either DMSO or various concentrations of LBH589. Twentyfour hours after treatment, cells were collected for gene expression analysis and washed with PBS. TRIzol reagent (Invitrogen) was used for total RNA isolation. cDNA was synthesized from isolated RNA using reverse transcriptase III (Invitrogen). Real time PCR primers were as follows: -actin, 5’-CACCAACTGGGACGACAT-3’ and 5’GCACAGCCTGGATAGCAAC-3’; NFκB1, 5’-ATTTGAAACACTGGAAGCACGA-3’ and 5’-GCGGATTAGCTCTTTTTCCCG-3’; RelA, 5’- GAATTCCAGTACCTGCCAGA-3’ and 5’-GGTCCGCTGAAAGGACTCTT-3’; CFLAR (encoding cFLIP), 5’-TTTCTTTGCCTCCATCTTGG-3’ and 5’GGGGGAGTTCGTCCTGTC-3’; CCND1 (encoding Cyclin D1), 5’GCGAGGACCAGAAGTGCGAGG-3’ and 5’-AGCTTGTTCACCAGGAGCAGC-3’; CDKN1A (encoding p21WAF1/CIP1), 5’-AGTCAGTTCCTTGTGGAGCC-3’ and 5’CATGGGTTCTGACGGACAT-3’. Reactions were run in triplicate on an iCycler iQ5 real time PCR detection system (Bio-Rad), using two-step SYBR green Supermix (BioRad), and results were analyzed with iQ5 software. The relative expression level of each gene was normalized to the β-actin expression level for each sample. Microarray analysis H295R (human adenocortical carcinoma), HeLa (human cervical cancer) and MCF-7her2 (HER2-overexpressed MCF-7) cells were treated with DMSO or LBH589 (50 nM) for 24 hours. For microarray analysis, total RNA was extracted using TRIzol reagent (Invitrogen). Synthesis and labeling of complementary RNA (cRNA) targets, hybridization of GeneChips, and signal detection were carried out by the Functional Genomics Core Facility at City of Hope. The Affymetrix Human Gene 1.0 ST Array (Affymetrix) was used for microarray gene expression profiles. The microarray was carried out using Ambion’s WT Expression kit (Life Technologies) and Affymetrix’s GeneChip Terminal labeling system. Briefly, 100 ng of total RNA was used to start the first strand cDNA synthesis using an engineered random primer plus polyT7 promoter. After the second strand cDNA synthesis, the antisense cRNA (in vitro transcription) was generated using T7 RNA polymerase. Then 10 μg of cRNA was used to start the second cycle of cDNA synthesis using random primers plus dUTP and dNTP. The single-strand cDNA was fragmented and then end-labeled with biotinylated nucleotides in the presence of terminal deoxynucleotidyl transferase (TdT) using Affymetrix’s WT Terminal Labeling kit. Labeled single-stranded cDNA (5 µg) was hybridized with an Affymetrix Human Gene 1.0 ST array, and the array was scanned using an Affymetrix GeneChip Scanner 3000 7G. Statistical processing of microarray data Microarray samples were RMA normalized [2] using Partek® Genomics SuiteTM (Version 6.5; Partek, Inc.), and genes were defined as differentially expressed if they showed at least a 1.5 fold-change and false discovery rate (FDR) < 0.05 (LBH vs. DMSO). FDR values were calculated using the method of Benjamini and Hochberg [3] from the distribution of ANOVA with Linear Contrast p-values. Our microarray data is MIAME compliant and the raw data has been deposited in NCBI Gene Expression Omnibus (GEO) with accession number of GSE36509. Systems-level analysis and statistical enrichment for groups/pathways listed in Ingenuity pathway analysis (IPA) (Ingenuity® Systems, www.ingenuity.com) were calculated using a Fisher’s Exact test. IPA was used to analyze genes that consistently showed up- or down-regulation following all 3 treatments (|fold-change| > 1.5, FDR < 0.05). Ingenuity Pathway Analysis IPA is a Web-based software program that identifies the biological functions, pathways, and mechanisms most relevant to a given data set of genes. Information on individual genes is drawn from a large knowledge base of biological networks created from millions of publications, and the networks are drawn by the Functional Analysis feature of IPA based on the connectivity of the genes. To obtain a comprehensive view of the differentially expressed genes, core analysis was performed, including network generation, functional analysis and canonical pathway analysis on the filtered (|fold change|>2, P < 0.05) genes. Clinical samples (Immunohistochemistry) Antigen retrieval was performed for 30 minutes in EDTA buffer at 98 oC. Sections were incubated for 30 minutes at room temperature using antibody (1/50 dilution). Slides were washed with Dako buffer, then incubated with a polymer secondary antibody (Envision + system; Dako), followed with the treatment of chromogen diaminobenzidine tetrahydrochloride (DAB; Dako), counterstained with hematoxylin, and mounted. Slides were scored by a pathologist who was blinded to the sample information, and were evaluated by light microscopy and scored as previously described [4]. First, a proportion ratio was assigned, which represented the estimated proportion of positive nuclear staining tumor cells. Next, an intensity score was assigned, which represented the average intensity of positive tumor cells (0, none; 1, weak, 2, intermediate; and 3, strong). The proportion and intensity scores were then multiplied to obtain a total score, which ranged from 0.6 to 2.85. Luciferase assay Cells were transiently transfected with the pNF-κB-luciferase expression construct (Stratagene) using the Lipofectamine Plus reagent system (Invitrogen) according to the manufacturer's protocol. Four hours post-transfection, cells were incubated for 24 hours before stimulation with TNFα (10 ng/ml) for one hour. Cell lysates were collected using passive lysis buffer and NF-κB activity assayed on a TD 20/20 luminometer (Turner Designs). Protein concentrations were assayed using the BCA Assay (Thermo Scientific). Data is expressed as relative luciferase units/protein content. NF-κB1 siRNA treatment siRNA oligonucleotides against NF-κB1 and control oligonucleotides (siControlA-A) were purchased from Santa Cruz Biotechnology. The siPORTTM NeoFXTM transfection reagent was purchased from Ambion Inc. AI-responsive and resistant cells were seeded in 6-well plastic tissue culture dishes in C/D FBS MEM at 1 x 105 cells/well. At various time points after siRNA treatment (7.5µg), cells were harvested for gene expression analysis. NF-κB1 transfection MCF-7aro cells were seeded (8 x 104 cells/well) in MEM with 10% C/D FBS in 12-well plastic tissue culture dishes. Cells were allowed to grow for 24 hours before transfection. Cells were cotransfected with 0.01 μg/well of pCMV4 p50 plasmid (Addgene). Mock transfection with only the Lipofectamine 2000 reagent (Invitrogen) was used as control. To determine whether NF-κB1 over-expressing MCF-7aro acquired AI-resistance, four hours post-transfection, cells were treated with 0, 50 or 100 nM letrozole for 2 and 4 days., after which the MTT assay was performed and NF-κB1 mRNA expression was measured. Animal studies (Establishment of LTET (long-term exemestane-treated) cell line from mouse xenograft) Briefly, one of the tumor specimens from the exemestane only-treated group was removed, digested with trypsin to make a single cell suspension, and cultured in MEM with Earle’s salts supplemented with 10% charcoal dextran-treated FBS, 1 mmol/l pyruvate, 100 units/ml penicillin, 0.1 g/l streptomycin, G418 (500 μg/ml), and 1 nM testosterone. 24 h 48 h 72 h 120 h 24 h 48 h 72 h 120 h Exe-R 1.5 1.0 24 h 48 h 72 h NF- B1 siRNA ** Control siRNA NF- B1 siRNA ** Control siRNA ** Control siRNA NF- B1 siRNA 0.0 ** NF- B1 siRNA 0.5 Control siRNA NF- B1 siRNA ** Control siRNA Control siRNA NF- B1 siRNA ** ** Control siRNA 0.0 * NF- B1 siRNA 0.5 Relative expression 1.0 NF- B1 siRNA NF- B1 siRNA ** Control siRNA Control siRNA NF- B1 siRNA ** ** Control siRNA NF- B1 siRNA 0.0 ** NF- B1 siRNA 0.5 LTEDaro 1.5 Control siRNA 1.0 Relative expression MCF-7aro 1.5 Control siRNA Relative expression Supplementary figures 120 h Supplementary Fig. 1. The expression of NF-κB1 was effectively suppressed by siRNA in all cell lines. siRNA-mediated knockdown of NF-κB1 in MCF-7aro, LTED, and Exe-R was confirmed by real-time PCR analysis 5 days after transfection. Cells were transfected with control siRNA or NF-κB1 siRNA. Gene expression was normalized to -actin. Columns, mean; bars, SE. *, p < 0.05; **, p < 0.01. Absorbance (570nm) (ratio to control) 1.5 1.0 0.5 0.0 0 1 5 10 20 LBH589 (nM) Supplementary Fig. 2. LBH589 is less effective in the suppression of MCF-10A, a noncancerous breast epithelial cells. MCF10A cells were treated with the indicated concentrations of LBH589 (LBH) or DMSO (vehicle control) for 6 days and medium was replaced every 72 h. Cell viability was assessed by MTT assay. Five replicates were performed for each measurement, and the mean and standard error were calculated. Data are shown as a ratio of treated samples to untreated controls. References 1. 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