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Cell Lines and Cultures

he various breast cancer cell lines (MCF-7, T-47D, SKBR3, MDA-435, and MDA-231) were maintained in DMEM containing 10% fetal bovineserum supplemented with glutamine and antibiotics.

SK-BR-3, MCF7, MDA-MB-435S, and MDA-MB-231 were obtained from the American Type Culture Collection. MDA-MB-435 was obtained from

National Cancer Institute. All cell lines were cultured in DMEM/F-12 (Cellgro Mediatech) supplemented with 10% (v/v) fetal bovine serum(SAFC Biosciences) at 37C and humidified in 5% CO2. For selected experiments, cells were treated with DMSO (Sigma) or rapamycin (LCLaboratories).

The cell lines to be used are SK-BR-3, MCF7, MDA-MB-435S, and MDA-MB-231. They are to

be obtained from the American Type Culture Collection. The cell lines were culture in

DMEM/F-12 from Cellgro Mediatech and supplemented with 10% (v/v) fetal bovine serum

at 37C and humidified in 5% CO2. The FBS also contains glutamine and antibiotics.

siRNA Synthesis, Labeling, and Transfection

Small interfering RNA (siRNA) to death domain-1 of integrin beta 3 binding protein is to be custom-ordered duplexes from Dharmacon or

Ambion. integrin beta 3 binding protein is also known as NRIF. Negative control siRNA were purchased. SilencersiRNA Labeling

Kit is to used according to manufacturer procedure in labeling siRNA. It employs Fluorescein dye.

Silencer siRNA tranfection Kit is to be used in transfection of the cell cultures.

Cytotoxicity Assays and Flow Cytometry

Typically, cell viability was assessed with a trypan blue exclusion assay as described in our previous publication (33). Apoptotic cell deathwas determined using an annexin V-FITC Apoptosis Detection kit (BD PharMingen, San Diego, CA) according to the manufacturer's manual.

Briefly, cells were harvested and washed with ice-cold PBS and then suspended in annexin V binding buffer. Then, cells were stained for 15minutes at room temperature in the dark and analyzed on a FACSCalibur flow cytometer using CELLQuest software. For clonogenic survivalassay, 103 cells were seeded in a 35 mm dish and transfected with the siRNAs as indicated in the figure legend. The media were changedevery 3 days and the cultures were observed daily for colony formation. On day 7, the cultures were washed with PBS, fixed, and stained aspreviously described (36). The colonies were counted under an inverted microscope.

mRNA Expression Analysis and Reverse Transcription-PCR

Total RNA was prepared using Trizol reagent (Invitrogen). To assess mRNA expression, a semiquantitative reverse transcription-PCR (RT-PCR) method was used as described previously (35). RT-PCR was done using a RETROscript kit (Ambion) per manufacturer's instructions.The primers and PCR conditions were described as follows: for human AR gene (forward 5-cctggcttccgcaacttacac-3; backward 5-ggacttgtgcatgcggtactca-3; adapted from ref. 6); human PSA gene (forward 5-gatgactccagccacgacct-3; backward 5-cacagacaccccatcctatc-3; ref. 37); and humanbcl-xl gene (forward 5-catggcagcagtaaagcaag-3; backward 5-gcattgttcccatagagttcc-3; ref. 38). 28S ribozyme RNA

(forward 5-gttcacccactaatagggaac gtg-3; backward 5-gattctgacttagaggcgttcagt-3) was used as an internal control. The primers weresynthesized by IDT. The amplification profile was as follows: 95C for 30 seconds, 56C for 30 seconds, and 72 C for 1 minute running in atotal of in 25 cycles. After 25 amplification cycles, the expected PCR products were size fractionated onto a 2% agarose gel and stained withethidium bromide.

RT-PCR and Western Blot Analysis.

Total RNA was isolated withTRIzol Reagent (Invitrogen). RT-PCR was carried out using the one-stepRT-PCR

system (Promega). Cell lysates from breast cancer cells were prepared after cell lysis in radioimmunoprecipitation

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assay buffer. Protein concentration was determined using the Bradford dye-binding assay (Bio-Rad).Eighty

micrograms of protein were electrophoresed on a 12% SDS-PAGE andtransferred to membrane. CXCR4 and actinproteins were detected using antiCXCR4 antibody (R&D Systems) and anti-actin antibody conjugated withhorseradish peroxidase (Santa Cruz Biotechnology), respectively, and chemiluminescence (enhanced

chemiluminescence; Amersham Pharmacia Biotech).Each experiment was repeated at least twice with similar

results.

Mitochondrial Membrane Potential and Caspase Activity

The siRNA-transfected cells were incubated in the presence of JC-1, which was added to the culture medium at a final concentration of 0.3g/mL for 15 minutes at 37C. Thereafter, the cells were analyzed under a fluorescent microscope. The caspase activity was measured usingan Apo-ONE Homogeneous Caspase-3/7 Assay kit obtained from Promega (Madison, WI) per the manufacturer's manual. Briefly, the cellswere washed in ice-cold PBS and then suspended in the assay buffer containing the substrate rhodamine 110 (Z-DEVD-R110) provided bythe supplier. The amount of fluorescent product generated is measured at 480/520 nM (wavelength) using a Fluoscan fluorescent reader asdescribed previously (32, 34).

Efficacy in Killing Cells That Survive Initial Treatment.

Noting that a small fraction of cells survive treatment with a cognate HCR transducer (between 1%and 5%in Fig. 3), we wished to establish

whether these surviving cells were resistant to HCR-mediated cell death. To examine this question, we transfected HCR transducers intocells grown up from the surviving populations. The cell counts of Fig. 4 reveal that HCR transducers mediate cell death with undiminishedefficacy in these regrown populations (20- to 100-fold population reductions). These results indicate that resistance to HCR-mediated celldeath is not responsible for the survival of a small fraction of cells following transfection with cognate HCR transducers.

Statistical Analysis

All experiments were repeated twice or thrice. Western blot results are presented from a representative experiment. The mean and SD fromtwo experiments for cell viability are shown. The number of viable/dying cells or cell colonies in the control group or the initial time pointwas assigned a relative value of 100%. The significant differences between groups were analyzed using the SPSS computer software (SPSS,Inc., Chicago, IL).