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2 protocols using hydroxypropyl methycellulose

1

Evaluating Combination Therapies on Ovarian Cancer Xenografts

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All animal work adhered to the Agency for Science, Technology and Research (A*STAR; Singapore), Institutional Animal Care and Use Committee (IACUC) guidelines on the use and handling of animals. SKOV3-Luc-D3 cells (Xenogen Co., Alameda, CA, USA) at a density of 3.5×106 in 100 μl of PBS were injected into the intraperitoneal cavity of 4-week-old female BALB/c nude mice. At 6 weeks post-implantation, the mice were randomly divided into control and treatment groups (n = 5 animals per group). For the treatment group, mice were administered via oral gavage with 50 mg/kg AZD0530, 50 mg/kg BIBF1120, or 25 mg/kg AZD0530 plus 25 mg/kg BIBF1120 (Selleck Chemicals, Houston, TX) for 5 days a week for 2 weeks. The drug was re-suspended in 0.5% hydroxypropyl methycellulose (Sigma-Aldrich) and 0.1% polysorbate buffer (Sigma-Aldrich). The control group received the vehicle buffer alone. The growth of tumor xenografts was monitored by bioluminescence using the IVIS system 2000 series (Xenogen Co.). The xenografts were harvested at 8 weeks post-implantation for gene expression microarray analysis (Affymetrix GeneChip Human Gene 1.0ST Array, Santa Clara, CA, USA) to ascertain the EMT scores, and then subjected to paraffin embedding followed by immunohistochemical staining for E-cadherin (#3195S, Cell Signaling Technology, Beverly, MA).
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2

Evaluation of PI3K/mTOR Inhibition

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Dulbecco’s modified Eagle medium (DMEM)/F12K was obtained from Invitrogen (Grand Island, NY) and RPMI medium from ATCC (Manassas, VA). Fetal bovine serum was purchased from Atlanta Biologicals (Lawrenceville, GA). The mTOR inhibitor rapamycin was obtained from Calbiochem (San Diego, CA). The Cell Death Detection ELISAplus kit was obtained from Roche (Indianapolis, IN). Annexin V Alexa Fluor 488 was purchased from Invitrogen (Grand Island, NY). The pBabe-puro p110α plasmids were obtained from Addgene (Cambridge, MA). Lipofectamine 2000 was acquired from Invitrogen (Grand Island, NY). NuPAGE 4–12% Bis-Tris gels were also purchased from Invitrogen (Grand Island, NY) and Sequi-blot PVDF membranes from Bio-Rad (Hercules, CA). The following antibodies were obtained from Cell Signaling (Beverly, MA): pAkt, Akt, pERK, ERK, p-p70S6K, p70S6K, pS6, S6, p4E-BP1, 4E-BP1, PARP, and cleaved caspase 3. An enhanced chemiluminescence (ECL) detection system was purchased from GE healthcare (Piscataway, NJ). The NT enzyme immunoassay (EIA) kit was acquired from Phoenix Pharmaceuticals (Belmont, CA). The serotonin ELISA kit was obtained from Immuno-Biological Laboratories Inc. (Minneapolis, MN, USA). 1-Methyl-2-pyrrolidinone (NMP), polyethylene glycol 300, hydroxypropyl methycellulose, and 0.2% Tween 80 were purchased from Sigma Aldrich (Saint Louis, MO).
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