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For the immunoprecipitation assay, cell lysates (600 μg) were im-munoprecipitated with protein A/G agarose beads (Merck Millipore, Temecula, CA, USA) which mixed with 1 μg of anti-acetyl-lysine anti-body in Fig. 3A, Hsp90 antibody in Fig. 3B and F-actin antibody in Fig. 5C, respectively. The precipitated beads were washed three times with 1 mL of ice-cold cell lysis buﬀer, after which the bound immune complexes were separated by SDS-PAGE and immunoblotting with the specific antibody.
3.6. HDAC fluorescence activity assay
Enzyme inhibition assays were conducted by the Reaction Biology Corporation (Malvern, PA, USA). The assay procedure has two steps . First, the fluorogenic substrate, which comprises an acetylated lysine side chain, is incubated with a purified HDAC enzyme. Deace-tylation of the substrate sensitizes the substrate so that, in the second step, treatment with the developer produces a fluorophore. Compounds were dissolved in DMSO and tested in at least 10-dose IC50 mode with 3-fold serial dilution starting at 10 μM. Trichostatin A was used as re-ference compound. The percentages of enzyme activity (relative to DMSO controls) and IC50 values were calculated using the GraphPad Prism 4 program based on a sigmoidal dose-response equation.
3.7. Wound healing assay
Cells were seeded in 6-well plates and cultured to 90% confluency, at which time the cellular monolayer was wounded with a sterile 10-μL pipette tip and washed with culture medium to remove detached cells. The cells were incubated in the presence or absence of test compounds for 24 h and photographed. The wound closure percentages were sub-sequently determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3.8. Immunocytochemistry and microscopy
MDA-MB-231 cells were cultured onto 15 mm cover glasses. After reaching 60% confluence, cells were treated with MPT0G211 or tu-bastatin A. Cells were then fixed with 4% paraformaldehyde for 10 min and washed with PBS for three times. Non-specific binding sites were blocked by incubation with 1% BSA in PBS containing 0.1% Triton X-100 and then probed with primary Midostaurin (PKC412) at 4 °C overnight. After three 10-min washes in PBS, the cells were stained for an additional 60 min with FITC-conjugated goat anti-mouse or anti-rabbit IgG
antibodies (1:100 dilution in PBS). DAPI (1 μg/mL) was used to stain nucleus. Subsequently, the samples were viewed and photographed using a TCS SP5 confocal laser-scanning microscope (Leica, Wetzlar, Germany) with appropriate fluorescence filters.
Removed orthotopic tumors were fixed in 4% paraformaldehyde in PBS. After fixation, the orthotopic tumors were embedded in paraﬃn and cut into 5 μm sections. The tumor sections were blocked with 5% BSA for 1 h and incubated with primary antibodies (1:100) in 0.5% BSA overnight. After three 10-min washes in PBS, the tumor sections were incubated for additional 60 min with HRP polymer conjugate reagent (Dako, Glostrup, Denmark). Immunohistochemical analyses were per-formed using DAB chromogen reagent (Dako, Glostrup, Denmark). Finally, the tissue sections were counterstained using hematoxylin and eosin.
The amounts of intracellular F-actin and G-actin were measured using the F-Actin/G-Actin In Vivo Assay Biochem Kit (Cytoskeleton, Denver, CO, USA). G-actin and F-actin were separated from protein samples obtained from cells and tissues according to the manufacturer's protocol. The total protein concentration in each tube containing either F-actin or G-actin was determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA), and the separated actin contents were analyzed by immunoblotting.
3.10. Metastatic assays in vivo
MDA-MB-231 cells (2 × 105 in 0.1 mL of medium) were injected into the tail veins of 5-week-old female severe combined im-munodeficient (SCID) mice under isoflurane anesthesia. Subsequently, MPT0G211 (25 mg/kg, qd), paclitaxel (10 mg/kg, q4d), or a combina-tion of MPT0G211 (25 mg/kg, qd) with paclitaxel (10 mg/kg, q4d) (all dissolved in a solution of 5% DMSO, 5% Cremophor, and 90% dextrose to 5% w/v) were administered intraperitoneally to the mice (n = 5 in each groups). At the experimental endpoint of day 73 post-tumor in-jection, the animals were sacrificed and the lungs were harvested for photography and immunohistochemistry.
All animal experiments were performed in accordance with the NIH guidelines on laboratory animal welfare and approved by the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine (IACUC number: 20110303).
Data are expressed as mean values ± standard errors of the means (SEM) and were analyzed using a one-way analysis of variance (ANOVA). If the ANOVA indicated significant intergroup diﬀerences, Tukey's post hoc test was used to determine the pairs of groups ex-hibiting these diﬀerences. A p value < 0.05 was considered statistically significant.