Cell proliferation assays

The CellTiter Glo Luminescent assay (Promega Corporation, Madison, WI, USA) was used to evaluate the relative cell proliferation of Ishikawa cells exposed to high and normal glucose conditions. The cells were seeded into 96-well plates at a density of 5,000 cells/well in 100 µl drug-free medium overnight. The cells were then treated with the indicated treatment (control, insulin, metformin or insulin plus metformin) for 72 h. After this period, the plates were equilibrated at room temperature for 30 min, and 100 µl of CellTiter Glo reagent was added in each well, mixed for 2 min and incubated at room temperature for 10 min. The luminescence was detected by a Centro LB 960 Microplate Luminometer (Berthold Technologies GmbH and Co. KG, Bad Wildbad, Germany).

In order to evaluate the sensitivity of the epithelial carcinoma cells to varying doses of metformin in high and normal glucose conditions, the Ishikawa cells were seeded into 96-well plates at a density of 5,000 cells/well in 100 µl drug-free medium overnight. The cells were then treated with increasing doses of metformin (0, 0.1, 1 and 5 mM) for 72 h. A CellTiter Glo Luminescent assay was performed as aforementioned. The effect of the different treatments was calculated as a fold-change compared with the control group. Each experiment was performed in sextuplicate and repeated three times.