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  • br Cell viability and cell proliferation assays br Cell viab


    3.3. Cell viability and cell proliferation assays
    Cell viability was measured using a colorimetric MTT assay. A total of 1 × 104 cells in 100 μL were placed in each well of a 96-well plate and incubated with vehicle (control) or vehicle plus test compound for 12 or 24 h. Subsequently, 1 mg/mL of MTT was added to the wells, followed by 2-h incubation at 37 °C. The cells were subsequently pel-leted and lysed in 100 μL of dimethyl sulfoxide, and the absorbance of each well at 550 nm was measured on a microplate reader.
    Cell proliferation was measured using the sulforhodamine B (SRB) assay. Cells (1 × 104) were incubated for 48 h with the indicated con-centrations (0, 0.3, 1, 3, 10, 30 μM) of test compounds, fixed with 10% trichloroacetic acid, stained for 30 min with SRB (0.4% in 1% acetic acid), and washed repeatedly with 1% acetic acid. Protein-bound dye was finally dissolved in 10 mM Tris Oxaliplatin solution, and the optical density at 510 nm was measured.
    TRIzol reagent (Invitrogen) was used to isolate total RNA from the cells. Single-strand cDNA was then synthesized from 5 μg of total RNA and used as a polymerase chain reaction (PCR) template. The following oligonucleotide primers were used for amplification: human aurora-A (GenBank Accession No. NM_198436), 5′-CAT CTT CCA GGA GGA CCA CT-3′ (forward) and 5′-CAA AGA ACT CCA AGG CTC CA-3′ (reverse); Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, GenBank Accession No. NM_002046), 5′-ATT CCA CCC ATG GCA AAT TC-3′ (forward) and 5′-TGG GAT TTC CAT TGA TGA CAA G-3′ (reverse). Real-time PCR was performed on a 7900HT Sequence Detection system (Applied Biosystems, Inc., Foster City, CA, USA). Quantitative gene expression levels were normalized to the expression levels of GAPDH in each sample. The relative mRNA expression level was determined by calculating the Ct values, and fold changes in expression were re-ported using the equation 2− Ct.
    3.5. Immunoblot and immunoprecipitation analyses
    (A) MDA-MB-231 cells (6 × 105) were incubated in 6-well plates until reaching 90% confluency, scratched with a pipette tip, and immediately photographed (0 h). The cells were incubated with or without MPT0G211 (1 and 10 μM, indicated as L and H, respectively), tubastatin A (10 μM), paclitaxel (0.01 μM), or an indicated combination treatment, allowed to migrate into the wound area for 24 h, and photographed. Cell migration into the wound was quantified using Image J software; the dashed line indicates time zero. The quantitative assessment of the mean number of cells in the denuded zone is expressed as the mean ± standard error of the mean from three independent experiments. (B) Cells were incubated with 10 μM of MPT0G211, tubastatin A or paclitaxel for 12 and 24 h, the cell viability were estimated by MTT assay. (C) The growth inhibitory evaluation (GI50, μM) of MPT0G211, tubastatin A, and SAHA in MDA-MB-231 and MCF-7 cells. *p < 0.05, **p < 0.01 compared with the control group; # p < 0.05, ## p < 0.01 compared with the indicated group.
    (caption on next page)
    Fig. 3. MPT0G211 treatment significantly inhibited aurora-A expression in human breast cancer cells. (A, B) MDA-MB-231 cells (1 × 106) were incubated for 8 h with or without MPT0G211 and tubastatin A (10 μM), and the total cell lysates were immunoprecipitated with antibodies against acetyl-lysine (A) or Hsp90 (B) and subjected to immunoblotting. (C, D) MDA-MB-231 and MCF-7 cells were treated with MPT0G211 or tubastatin A (1 and 10 μM, respectively) for 24 h, after which aurora-A mRNA levels were measured using real-time PCR (C); total cell lysates were prepared for a western blot analysis of the indicated proteins (D). (E) MDA-MB-231 and MCF-7 cells were incubated with MG132 (3 μM) for 30 min prior to treatment with MPT0G211 or tubastatin A (10 μM) for another 24 h. Cell lysates were subjected to western blot analysis using the indicated antibodies. (F) Cells were transfected with 1 μg of a vector encoding HDAC6-Flag for 24 h, and then treated with MPT0G211 or tubastatin A (10 μM) for another 24 h. Cell lysates were collected to detect the expression levels of indicated proteins. The results in (A-F) are the means ± standard errors of the means from three independent experiments. *p < 0.05, **p < 0.01 compared with the control group; # p < 0.05, ## p < 0.01 compared with the indicated group.
    Signaling Technology, Danvers, MA, USA), scraped from the culture surface, incubated on ice for an additional 10 min, and centrifuged for 30 min at 17,000 g and 4 °C. Protein samples (20 μg) were then elec-trophoresed on sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred onto nitrocellulose membranes, which were blocked by incubation with 5% fat-free milk in phosphate-buffered saline (PBS) for 30 min at room temperature. Immunoblotting was performed by incubating the membranes with primary antibodies in PBS overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. To measure bound antibodies, the membranes were treated with an enhanced chemilu-minescence reagent (Advansta Corp., Menlo Park, CA, USA) and ex-posed to photographic film.