Temsirolimus

The mTOR inhibitor temsirolimus was purchased from Pfizer (New York, NY, USA) and was dissolved in its accompanying diluent to a stock concentration of 10 mM and stored at –20°C. This stock solution was diluted to reach a final concentration of 0.01–10 nM prior to use with culture media, and the cells were exposed to the drug for 48 h prior to clonogenic assays and western blot analyses.

Normal human smooth muscle (HA-VSMC), RMS (RH30, aRMS), (RD, eRMS) and rhabdoid cell line (A204) cells were procured from American Type Culture Collection (ATCC). While (CW9019, aRMS) and (SMS-CTR, eRMS) cells were gifted to JGP by Dr. Frederic G. Barr, Deputy Branch Chief and Senior Investigator Laboratory of Pathology, National Cancer Institute, USA. RMS cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 μg/ml of streptomycin at 37°C in a humidified atmosphere of 5% CO₂. HA-VSMC were grown in F-12K medium supplemented with 0.05 mg/ml ascorbic acid, 0.01 mg/ml insulin, 0.01 mg/ml transferrin, 10 ng/ml sodium selenite, 0.03 mg/ml endothelial cell growth supplement (ECGS); fetal bovine serum to a final concentration of 10%, HEPES to a final concentration of 10 mM, TES to a final concentration of 10 mM. GANT-61 was purchased from Cayman (MI, USA). Rapamycin was purchased from Fujian Kerui Pharmaceutical Co., Ltd (Fuzhou, Fujian, China). Temsirolimus and vincristine were obtained from Pfizer (NY, USA) and Hospira (IL, USA) respectively. Primers used in this study were obtained from Invitrogen (Supplementary Table S1-I and II). Primary antibodies were purchased as described in supplementary Table S2. Various treatments of these cells were performed when their confluence reached to about 70-80%.

COTI-2, supplied by Cotinga Pharmaceuticals (London, Ontario), was dissolved in 100% dimethyl sulfoxide (DMSO) stock solution and diluted in αMEM or DMEM plus 10% FBS so that final DMSO concentrations were 0.5–1.0%. For the AN3-CA xenograft studies, 235 mg of COTI-2 was mixed with 4.84 g of Captisol® (CydexPharmaceuticals, Lenexa, KS) in 10 ml of sterile water (Hospira, Inc., Lake Forest, IL). For the PANC-1 xenograft studies, COTI-2 was dissolved in phosphate-citrate buffer at pH 2.3.
Cisplatin, 5-FUdR (Sigma-Aldrich, St. Louis, MO), vinorelbine (GlaxoSmithKline, Brentford, UK), temsirolimus, rapamycin (Pfizer, New York, NY), gemcitabine (Eli Lilly, Indianapolis, IN), paclitaxel, carboplatin (Bristol-Myers Squibb Co., New York, NY), and vincristine were obtained from the London Health Sciences Centre Pharmacy (London Regional Cancer Program, London, Ontario). Cetuximab was purchased from ImClone Systems (New York, NY). Erlotinib was obtained from LC Laboratories (Woburn, MA).