Jang et al. BMC Cancer 2010, 10:391 http://www.biomedcentral.com/1471-2407/10/391 glycerol, 14.4 mmol/L 2-ME, and 0.0002% (w/v) BPB], and subjected to SDS-PAGE and Western blotting. Western blotting For western blot analyses, cells were harvested after 24 or 48 hours of transfection and lysed with lysis buffer (5 mM/L ethylenediamine tetra acetic acid; 300 mM/L NaCl; 0.1% NP-40; 0.5 mM/L NaF; 0.5 mM/L Na3VO4; 0.5 mM/L phenylmethylsulfonyl fluoride; and 10 μg/ml each of aprotinin, pepstatin, and leupeptin; Sigma, St Louis, MO). Following centrifugation at 15,000 g for 30 minutes, the concentrations of supernatant proteins were analyzed using the Bradford reagent (Bio-Rad, Her- cules, CA). For analysis of protein contents, 50 μg of total proteins was electrophoresed in 10% SDS-PAGE gel, and transferred to polyvinylidene difluoride mem- branes (Millipore, Bedford, MA), which were then incu- bated with the respective antibodies indicated above. Immunoblots were visualized using an enhanced chemi- luminescence detection system (Amersham Pharmacia Biotech, Uppsala, Sweden). Reverse transcription-polymerase chain reaction (RT-PCR) Cells were collected after 24 hours of transfection. Total RNA was extracted using Trizol (Invitrogen), according to the manufacturer’s instructions. For RT-PCR analysis, 5 μg total RNA was reverse-transcribed using RT-PCR kits (Pro- mega, Madison, WI). PCR was used for amplification of target cDNA under the following conditions: 35 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 min- utes. PCR products were analyzed using standard agarose gel electrophoresis. The primer sequences were used as fol- lows: for VEGF, forward 5′ CTACCTCCACCATGC- CAAGT 3′ and reverse 5′ GCAGTAGCTGCGCTGCG CTGATAGA 3′; for MT1-MMP, forward 5′ CCATTGGG- CATCCAGAAGAGAGC 3′ and reverse 5′ GGATACC- CAATGCCCATTGGCCA 3′; for MMP9, forward 5′ TT CATCTTCCAAGGCCAATC 3′ and reverse 5′ CTTG TCGCTGTCAAAGTTCG 3′; for MMP2, forward 5′ GGCTGGTCAGTGGCTTGGGGTA 3′ and reverse 5′ AGATCTTCTTCTTCAAGGACCGGTT 3′; and for GAPDH: forward 5′ ACCACAGTCCATGCCATCAC 3′ and reverse 5′ TCCACCACCCTGTTGCTGT 3′. Fluorescence-activated cell sorter analysis (intracellular and surface staining) pSilencer™ 3.1-H1 puro ANT2 siRNA vectors as well as pSilencer™ 3.1-H1 puro scramble siRNA vectors were transfected into SK-BR3 cells for the indicated times. Six hours before harvesting, the cells were treated with brefeldin A (10 μg/ml), washed twice with PBS, fixed with 2% paraformaldehyde, and permeabilized with buf- fer (1% BSA, 0.1% saponine, 0.1% sodium azide in PBS), followed by staining with phenylethylene-conjugated Page 3 of 9 anti-VEGF as well as anti-mouse IgG antibodies (BD Pharmingen, San Diego, CA) for 1 hour at 4°C and ana- lysis by flow cytometry (Epics XL; Coulter). Gelatin zymography Conditioned medium and cell lysates were electrophor- esed in a polyacrylamide gel containing 1 mg/ml of gela- tin. As previously described, proteolysis was detected as a white zone in a dark blue field [29]. Invasion and migration assays Matrigel invasion assays were performed using modified Boyden chambers with polycarbonate Nucleopore mem- branes (Corning, Corning, NY). Precoated filters (6.5 mm in diameter, 8 μm pore size, Matrigel 100 μg/cm2) were rehydrated with 100 μL medium; 2 × 105 cells in 200 μL serum-free DMEM supplemented with 0.1% bovine serum albumin were seeded into the upper part of each chamber. Following incubation for 18 hours at 37°C, noninvaded cells on the upper surface of the filter were wiped out with a cotton swab; and the invaded cells on the lower surface of the filter were fixed and stained using a Diff-Quick kit (Fisher Scientific, Pitts- burgh, PA). Invasiveness was determined by counting cells in five microscopic fields per well, and the extent of invasion was expressed as average number of cells per microscopic field. Transwell migration assays were performed using the same procedure as that used in performance of the invasion assay, except that underside filters with type I collagen was coated. Results ANT2 shRNA induces dissociation of HER2/neu from HSP90, followed by proteasomal degradation of HER2/ neu in breast cancer cells We first examined regulation of HER2/neu by ANT2 shRNA in HER2/neu-overexpressing breast cancer cell lines, SK-BR3. As shown Fig. 1A, HER2/neu was down- regulated by ANT2 shRNA transfection for 24 hours. At that time, ANT2 shRNA had little influence on the cell viability by MTT assay, although the morphology of cells seemed not to be the best under microscopic examination. Co-immunoprecipitation of HSP90 and HER2/neu revealed that knockdown of ANT2 by shRNA resulted in effective dissociation of HER2/neu from HSP90, and concomitant increased poly-ubiquiti- nation and degradation of HER2/neu protein (Fig. 1B and 1C). We used HSP90 inhibitor, 17-AAG, as a posi- tive control, and the levels of ANT2 shRNA-mediated dissociation from HSP90 and down-regulation of HER2/ neu protein were nearly comparable to those observed in 17-AAG-treated cells. To address the mechanism to inhibit HSP90 activity, we measured the ATP level in ANT2 shRNA-transfected cells. As observed in other