Cancer cells were seeded in 96-well plates in triplicate at a density of 3,000 cells per well, and kept overnight to adhere. The cells were then treated with five different concentrations of inhibitors in the presence of medium with FBS in triplicate at 37°C and put into either normoxic (O2=21%) or hypoxic (O2=1%) incubators in parallel for 48 h. Additionally, cells seeded in 96-well plates (3,000 cells/well) were transfected with a small interfering RNA for STAT3 (STAT3 siRNA; Cell Signaling Technology, Beverly, MA, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in triplicate, according to the manufacturer’s protocol for 48 h, and then cultured under either normoxic or hypoxic conditions for 24 h.
Twenty-five milliliters of Thiazolyl Blue Tetrazolium (Sigma, St. Louis, MO, USA) was added to each sample and plates were further incubated for 4 hours. After that, 100 μl of N,N-dimethylformamide (Sigma) solubilizing agent was added to each well and plates were placed on a shaker overnight at room temperature. On the following day, the absorbance was read at 595 nm. IC50 against cancer cell lines were calculated for LLL12 and LY5 using GraphPad Prism 7 (GraphPad Software, Inc.).
Twenty-five milliliters of Thiazolyl Blue Tetrazolium (Sigma, St. Louis, MO, USA) was added to each sample and plates were further incubated for 4 hours. After that, 100 μl of N,N-dimethylformamide (Sigma) solubilizing agent was added to each well and plates were placed on a shaker overnight at room temperature. On the following day, the absorbance was read at 595 nm. IC50 against cancer cell lines were calculated for LLL12 and LY5 using GraphPad Prism 7 (GraphPad Software, Inc.).