Azd2014

Following transplantation of PDX tumors, mice were randomly assigned to treatment groups and monitored for tumor growth by twice-weekly palpation. Once tumors had reached a size of 5 mm × 5 mm (~65 mm³) treatment was initiated. Dasatinib (10 mg/kg) and AZD2014 (20 mg/kg – MedChem Express, HY-15247) were formulated in 30% Sulfobutylether-β-cyclodextrin (MedChem Express, HY-17031) in water and administered by daily oral gavage. Mice were weighed twice weekly and doses were adjusted according to bodyweight. Tumor growth was measured by twice-weekly caliper measurements. Drug administration, data collection and data analysis were performed by separate individuals who were blinded with respect to the treatment group of each mouse.

All chemicals and reagents in this work were purchased and used without further treatment or purification. General laboratory reagents were purchased from Sigma-Aldrich (UK) unless otherwise stated. AZD2014 and INK128 were purchased from MedChem Express (Europe). Cell culture dishes (35 mm × 20 mm) were bought from MatTek (USA) and 96-well round plates from (Corning™ Falcon™). Propidium iodide was purchased from Sigma-Aldrich (UK). ER-Tracker™ Red and LysoTracker Red DND-99 was purchased from Thermofisher Scientific (UK). mDsRed-Rheb, EGFP-Rheb, EGFP-mTOR and EGFP-S6K1 DNA plasmids made as previously described [14 (link)] [45 (link)]. EGFP-raptor plasmid was a kind gift from Prof. Jacek Jaworski (Poland) and purified GFP was a kind gift from Prof. Cameron Naylon (Australia).
All cell culture reagents were purchased from Thermofisher Scientific (Gibco™).
Human Embryonic Kidney (HEK293) cells were purchased from ATCC (U.S.A.), certified as mycoplasma (contamination) free. Michigan Cancer Foundation-7 (MCF-7) breast cancer and Chinese Hamster Ovary (CHO) cells were initially purchased from ECACC (UK) and mycoplasma free.