Gen5 1

The HepG2 and Huh7 cells were transfected with a plasmid coding for the luciferase gene (pCDNA3.1-Luc) using the jetPEI® reagent (Polyplus-transfection®, Illkirch, France) according to the protocol of the manufacturer. The plasmid coding for the luciferase gene (pCDNA3.1-Luc) was donated by Dr J. Massagué (Memorial Sloan-Kettering Cancer Center, New York, NY, USA). Positive transfected cells were obtained subsequent to selection with the appropriate antibiotic, 450 µg/ml hygromycin. Once the clones had been isolated they were independently harvested by cloning rings, grown in multiwell plates and expanded. Hygromycin positive clones were tested for the presence of the luciferase gene by adding 150 µg/ml luciferin to the culture media, and then reading the luminescence in triplicate wells using a Synergy4 multi-mode microplate reader with Gen5 1.05 software (BioTek Instruments, Inc., Winooski, VT, USA). Each clone was analyzed at least twice and one of them, HepG2-Luc#22, was chosen for further experiments. In addition, a previously published MCF7-lucifesase expressing clone was established as previously described (22 (link)) and analyzed in parallel as a positive control. The correct behavior of these clones in terms of general aspect, proliferation (measured as MTT metabolization) and cell cycle profile was determined before further proceeding.

For cell cycle analysis by flow cytometry, ethanol-fixed cells were stained with 5 μg/ml propidium iodide (PI) and 250 μg DNase-free RNAse. A total of 50,000 cells were acquired in the PI gate by using a BD Accuri™ C6 flow cytometer and the C6 (version 1.0.264.21) software (BD Biosciences).
To measure bromodeoxyuridine (BrDU) incorporation, the Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Diagnostics GmbH, Mannheim, Germany) was used following the manufacturer's instructions. Data were acquired using a BioTek Synergy 4 reader with Gen5 1.05 software (both from BioTek Instruments, Inc., Winooski, VT, USA).

K562/A were cultured as aforementioned and treated with 200 nM BEZ235 for 24 h. Total protein was extracted from K562/A cells using lysis buffer (1% Triton X-100; 150 mM NaCl; 2 mM EDTA; 50 mM Tris-HCl) supplemented with phosphatase inhibitor cocktail. Total protein concentration was measured using Gen5 1.0 software (BioTek Instruments, Inc.) on a spectrophotometer microplate reader (BioTek Instruments, Inc.). A total of 20–50 µg protein was separated by SDS-PAGE using 10–15% gels and transferred onto a PVDF membrane. Following blocking non-specific binding sites with 5% non-fat dry milk in Tris-buffered saline with 0.05% Tween-20 at room temperature for 60 min, the membranes were incubated with primary antibodies at 4°C overnight. Next, the membranes were probed with HRP-conjugated secondary antibody (1:5,000) for 60 min at 37°C. Subsequently, the immunoreactive membranes were developed using Pierce™ Electrochemiluminescent Western Bloting Substrate (cat. no. UC280185, Thermo Fisher Scientific, Inc.) on ImageQuant™ LAS 4000 (GE Healthcare Life Sciences) and the density was quantified and normalized to β-actin.

Total cell proteins were extracted from K562 cells treated with rapamycin and/or doxorubicin using lysis buffer (1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl) supplemented with phosphatase inhibitor cocktail. After protein concentration was measured by Gen5 1.0 software (BioTek Instruments, Inc.) on a spectrophotometer microplate reader (BioTek Instruments, Inc.), 20–40 µg protein was run on a 10–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to a polyvinylidinedifluoride membrane. Following blocking non-specific binding sites with 5% nonfat dry milk in tris-buffered saline with 0.05% Tween at 37°C for 60 min, the membranes were incubated with primary antibodies against mTOR, p-mTOR, p70S6K, p-p70S6K, Bcl-2, Bax, Bcl-xL, CDK4, CDK6, Cyclin B1 and CyclinD1at 4°C overnight. Next, the membranes were probed with horseradish peroxidase-conjugated secondary antibody (1:5,000) for 60 min at 37°C. Subsequently, the immunoreactive membranes were developed using Pierce™ ECL Western Blotting Substrate (cat. no. 32109; Thermo Fisher Scientific, Inc. cat. no.32109) on Luminescent Image Analyzer (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Band density was quantified and normalized to β-actinby Image J software (version 2; National Institutes of Health).