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Blood, Vol. 113, Issue 22, 5526-5535, May 28, 2009
Alcohol consumption and decreased risk of non-Hodgkin lymphoma: role of mTOR dysfunction
Blood Hagner et al.
113: 5526
Supplemental materials for: Hagner et al
Cell culture and treatment
Burkitt’s Lymphoma (Raji) cells and diffuse large B-cell lymphoma (SUDHL-4) cells were cultured in RPMI-1640 containing 10% fetal bovine serum and 1% Penicillin/Streptomycin. Mammary epithelial adenocarcinoma (MCF-7, BT-474, and BT-20) cells were cultured in Dulbecco’s modified essential medium containing 10% fetal bovine serum and 1% Penicillin/Streptomycin. Cells were treated with ethanol concentrations (0–20 mΜ) for 10 days, 10 nΜ rapamycin or 40 µM compound C (Calbiochem) for 24 hours. Cells were cultured in a closed flask system, and centrifuged every other day with ethanol replenished in fresh media to prevent effects from evaporation. A minimum of three independent experiments was performed. Viral transduction of mTOR constructs
Lentivirus vectors encoding vector, wild-type mTOR (wt-mTOR) and rapamycin-resistant mTOR mutant (rr-mTOR), containing an SI substitution in the FKBP12-rapamycin binding domain, were kindly provided by J. Silvio Gutkind. Transfections in 293T virus packaging cells and subsequent transduction were conducted as previously described.1 REFERENCE 1. Sodhi A, Chaisuparat R, Hu J, Ramsdell AK, Manning BD, Sausville EA, et al. The TSC2/mTOR pathway drives endothelial cell transformation induced by the Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor. Cancer Cell. 2006;10:133–43.
Files in this Data Supplement:
- Figure S1. Ethanol inhibits mTOR signaling in a rapamycin independent and AMPK independent process (JPG, 182 KB)
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(A) SUDHL-4 cells, separated into three treatment groups (0 mΜ, 20 mΜ or Rap.), were transduced with either vector, wild type mTOR (wt-mTOR) or a rapamycin resistant mTOR (rr-mTOR). 50 µg of total protein lysates was loaded and the abundance of pmTOR, p-p70S6K, and β-actin was assessed. (B) Raji, and SUDHL-4 cells were treated with either ethanol (0–20 mΜ), compound C (40 µM) or amino acid starved media. A unit of 50 µg of total protein lysates was loaded and the abundance of pAMPK and β-actin was assessed.
- Figure S2. Chronic ethanol treatment does not affect viability of lymphoma cells (JPG, 281 KB)
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Raji cells were treated with ethanol (0–20 mΜ) for 10 days and analyzed by Annexin/PI staining to identify the apoptotic population. Forward scatter plot of cells, apoptotic population (area in upper right quadrant) was measured via Annexin V and Propidium Iodide (PI). There was minimal difference in apoptosis induction between control and treated cells.
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