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Novanor 1 levonorgestrel 1.5 mg Pregnyl 1.5 mg Norinyl 1.5 mg Ethinyl 1.5 mg E2 1.0 mg Estradiol 1.0 mg Nortestosterone 2.5 mcg Oestradiol 2.5 mcg Progesterone 1.0 mg Ethylestradiol 1.0 mg Aldosterone 0.25 mg Androstenedione 0.2 mg AUC 100 Sibutramine 15mg 60 pills US$ 280.00 US$ 4.67 : 0-5 h AUC 100 : 6-14 h AUC 50 : 2-3 h AUC 50 : 4-6 h In vitro, C3H cells were seeded at a density of 3×105 Cells/cm2 in 24-well plates, treated with a vehicle control (DMEM/F12 medium with 0.1% FBS) or E2 for 5 days. Then, the cells were treated with different mixtures of 1% estradiol, and 2% levonorgestrel, 5% 2.5% 0.2, canada drug pharmacy discount codes and 0.8% noredron, 1, 4% ethinyl estradiol on a weekly schedule. In all treatments, an additional dose of 1.0 mg (E2, 1% and 2%) was administered before the next dose of E2. All other chemicals were chosen according to in vitro testing data. All chemical combinations were used at a minimum concentration of 0.2%, and the same doses were administered. For a better comparability of the treatment C3H cells, and to improve the accuracy of results, treatment protocols were repeated three times with a minimum concentration of 0.4%. All the above chemical combinations were used in C3H cell culture at a minimum concentration of 0.8%. Cell proliferation was tested for 3 consecutive days, starting on the first day of injection. data were analyzed on a Student's t test after adjustment for multiple testing. Cell Proliferation Measurements Cells used as controls were grown in a serum-free medium at 37 °C. Cells were lysed in lysis buffer (Tris-HCl: Tris [pH 7.4], 150 mM NaCl, 3 EDTA, protease inhibitor cocktail 1 [Peprotech, UK]), 2 mg/ml Na-GTP (Sigma-Aldrich, Germany) and 0.1% SDS. Proteins were precipitated with 0.1 N Tris-HCl; 150 mM NaCl; 20 sodium pyrophosphate; 100 mM phosphoric acid; and 0.4% Triton X-100. Dried membranes were probed with 2 μg/ml pTriton X-100 (Sigma-Aldrich, Germany) prior to analysis according the manufacturer's protocol. Statistical analysis All tests were performed with Prism version 5 (GraphPad Software). Acknowledgments This work was supported by the Italian Ministry of University and Research (PRR 5/03/14-DOG-SITO) under the project "Determinanti, Sesteti, Medici" under Grant Agreement No. 2012UCT1-2326. Author Contributions Conceived and designed the experiments: CRB. Performed Analyzed data: Contributed reagents/materials/analysis tools: CRB. Wrote the paper: CRB LMM. All authors gave the final approval in writing. Conflict of Interest Statement The authors declare that research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Acknowledgments This work was partly supported by The Italian Society for Clinical Pharmacology Grants, FP7814 and DP072, by Italian National Research Council and European Funding Grant no. MNRANB013078, FP72412, EP062359–FP072501, PCT/GB2009-003027‐G and PCT/GP2009-010357.
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