Fig. 3

Growth-inhibitory effects of metformin in BCAHC-1 cells. A Cell cycle analysis performed by flow cytometry in BCAHC-1 cells treated with vehicle or 10 nM insulin for 6 h alone or in combination with 2 mM metformin, which was added to culture medium 18 h before the treatment with vehicle or insulin. B Percentage of BCAHC-1 cells in G0/G1, S and G2/M phases of cell cycle. The values represent the mean ± SD of three independent experiments. C Proliferation of BCAHC-1 cells after 5 days treatment with vehicle or 10 nM insulin alone or in combination with 2 mM metformin (Met), which was added to culture medium 18 h before the treatment with vehicle or insulin. Values of cells treated with vehicle were set as 100% upon which proliferation induced by treatments was determined. D Colony formation assay in BCAHC-1 cells exposed to vehicle or 10 nM insulin alone or in combination with 2 mM metformin, which was added to culture medium 18 h before the treatment with vehicle or insulin. The plates were stained with Giemsa and colonies were counted following 10 days of incubation (E). F Representative pictures of spheroids (a single spheroid/well) grown on agar-coated plates upon 20 days treatment with vehicle or 10 nM insulin alone or in combination with 2 mM metformin. Scale bar: 100 μm. G Quantification of BCAHC-1 spheroid growth. The number of cells treated with vehicle was set as 100% upon which the number of cells upon treatments was determined. Each data point is the mean ± SD of three independent experiments performed in triplicate. (*) indicates significant differences with respect to vehicle sample (p < 0.05); (black square) indicates significant differences with respect to Insulin treated sample (p < 0.05); (white circle) indicates significant differences with respect to Metformin treated sample (p < 0.05)