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  • br Fig AKBA significantly inhibited tumor growth in an


    Fig. 5. AKBA significantly inhibited tumor growth in an A2780/Taxol xenograft model. (A–C) Representative images of tumors and tumor growth curve after cell inoculation and treatment with AKBA in an A2780/Taxol nude mouse xenograft model (Mean ± SD, n = 6, **P < 0.01 versus model group). (D) Drug-resistant related proteins P-gp, LRP, BCRP and MRP in xenografts were analyzed by western blotting and the results of density quantification were shown using GAPDH for normalization (Mean ± SD, n = 3, **P < 0.01 versus model group).
    A2780 and A2780/Taxol GW3965 were incubated in 6-well plates and treated with AKBA for 24 h. 5 μmol/L Rhodamine 123 (Sigma, St Louis, MO) was added and incubated for another 2 h in dark. Cells were then collected and washed twice with ice-cold phosphate-buffered saline (PBS). Accumulation of intracellular Rhodamine 123 was analyzed by FACS scan flow cytometry (Becton Dickinson, San Jose, CA).
    2.5. Cell apoptosis analysis
    A2780/Taxol cells (5 × 105) were collected after trypsinization, wash and centrifugation. Cells were resuspended in 500 μL binding buffer containing 5 μL Annexin V-EGFP and 5 μL of PI. Fluorescence of samples was detected and analyzed using FCM after 10 min incubation in dark. 
    2.6. Cell cycle analysis
    After treatment with AKBA for 72 h, A2780/TAXOL cells (5 × 105) were harvested and washed twice with PBS, resuspended and fixed with cold 75% ethanol at 4℃ overnight. The fixed cells were washed with PBS, then incubated with 100 μL RNase A at 37℃ for 30 min followed by staining with 400 μL propidium iodide (PI) solution in dark. Cell cycle was analyzed on FACS scan flow cytometry.
    2.7. Wound healing assay
    Cells were cultured in six-well plates and scratched with 1 mL pip-ette tips followed by washing with PBS to remove any cellular debris. AKBA was added into wells for 48 h incubation, and the wounds were photographed using Olympus IX71 microscope (Olympus Corporation, Tokyo, Japan) at h and 48 h. The extent of wound closure was mea-sured for the wound areas obtained from five independent fields using Image Pro Plus 5.1.
    2.8. Cell invasion assay
    The invasion assay was conducted using transwell chambers (24-well, 8.0-μm pore membranes; Costar, Cambridge, MA, USA). 50 μL of 5.0 mg/ml Matrigel basement membrane matrix (BD Biosciences) was added to upper chambers and allowed to gel for 30 min at 37 °C. Cells (5 × 105) in 200 μL serum-free RPMI 1640 were seeded into upper chamber and medium containing 10% FBS was added to lower chamber. After being incubated at 37 °C for 24 h, noninvasive cells from the upper chamber were completely removed using cotton swabs. The invasive cells migrated to the lower membrane surface were fixed with paraformaldehyde and stained with Crystal Violet for 10 min. The number of invasive cells was counted with a microscope.
    2.9. Western blotting
    Total cellular and tissular protein extracts were prepared in RIPA lysis buffer supplement with 10 mM PMSF. Protein concentrations were determined using the BCA protein assay kit (Enjing Biotech, Nanjing, China). 40 μg proteins were separated using 10% SDS-PAGE and transferred to GW3965 PVDF membrane (Millipore, Bedford, MD, USA). The membranes were blocked with 5% skimmed milk and incubated with the P-gp, LRP, BCRP, MRP, GAPDH primary antibodies and HRP- con-jugated goat anti-rabbit secondary antibodies (EnoGene, New York, USA), further visualized using the enhanced chemiluminescence (ECL) procedure. The grey density of target bands was analyzed by Image J software (National Institutes of Health, Bethesda, MD, USA) and nor-malized to GAPDH.
    Athymic nude mice (BALB/c-nu, female), 4 weeks old and weighing 14–16 g, were purchased from Nanjing Model Animal Research Center (MARC). All animals were provided sterilized food and water. All ex-periments were carried out in accordance with the guidelines on animal care and experiments of laboratory animals, which was approved by the ethics committee for animal experiments.