Supplies and Tactics two.one. CellCulture. Human ovarian carcinoma cell lines, SKOV- three and OVCAR-3 , and breast carcinoma cell lines, SKBR-3 and BT-474 , had been obtained from your American Style Culture Collection . The MCF-7/HER2 human breast carcinoma cell line was kindly supplied by Dr. M. C. Hung . The MDA-MB-435/HER2 human melanoma cell line was kindly offered by Dr. T. D. Way . All cells had been cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum in a humidified ambiance of 5% CO2 at 37?C. 2.2. Chemical compounds and Antibodies. Thethiazolyl blue tetrazolium bromide , cycloheximide , and N-acetyl-Lleucinyl- L-leucinyl-norleucinal were obtained from Sigma-Aldrich . Antibodies towards cyclins D1 and E, p21, p27, phospho-Akt , Akt1, and ubiquitin had been bought from Santa Cruz Biotechnology, Inc. . Antibodies towards phospho-PI3K, PI3K, phospho-Erk 1/2, and Erk 1/2 had been obtained from Cell Signaling Engineering, Inc. .
Antibodies towards phospho-HER2 , HER2 , ??-actin, and Ki-67 have been purchased C59 wnt inhibitor clinical trial from Neomarkers Inc. , Calbiochem , Chemicon Global Inc. , and Dakocytomation Inc. , respectively. Taxol was purchased from Bristol-Myers Squibb , and cisplatin was purchased fromPharmacia & Upjohn S.p.A. . two.3. Preparation of Ganoderma tsugae Extracts. Ganoderma tsugae was kindly offered by the Luo-Gui-Ying Fungi Agriculture Farm , Taoyuan, Taiwan. The extract of GT was prepared as described previously . Briefly, the powder of the GT fruiting body was soaked in 99.9% methanol , mixed, and shaken for 24 h on a rotating shaker. After centrifugation, the supernatant was poured through filter paper , and the residues have been extracted with methanol two additional times as mentioned above.
The filtrates were collected together and subjected to concentration under reduced pressure to produce a brown gel-like Raf Inhibitors GT extract . The yield was approximately 30%. The GTE was then prepared as a stock solution with methanol solvent and stored at ?80?C until use. For animal experiments, the dry GTE was redissolved in ethanol and diluted with a suspension solution to a concentration of 10mg/mL. two.4. Quality Control of GTEs via Bioresponse Fingerprinting. The quality of the GTEs was assessed as described previously . Briefly, the genomic bioresponse to the GTEs was determined in SKOV-3 cells treated with 0.5mg/mL of GTE. The total RNA was extracted from the GTE-treated cells, cleaned with a commercial kit , and then used to obtain transcription profiles in GeneChip hybridization studies using Affymetrix engineering.
The changes in the individual gene expression levels obtained by the GeneChip experiments had been measured by Affymetrix MAS 5.0 software. A statistical pattern comparisonmethod from the PhytomicsQC platform, Phytomics Similarity Index , was applied to determine the batchto- batch similarity of the botanical products. In general, clinically similar batches have a PSI more than 0.95. two.5.