Fig. 2

Metformin abrogates c-Fos and Cyclin D1 expression induced by insulin in BCAHC-1 cells. A Immunoblots of c-Fos from BCAHC-1 cells treated for 6 h with vehicle (−) or 10 nM insulin (Ins) in the presence or absence of 1 µM IR inhibitor OSI-906, 1 µM PI3K inhibitor alpelisib or 100 nM MEK inhibitor trametinib. B BCAHC-1 cells were transiently transfected for 18 h with a c-Fos promoter construct, then were treated for 12 h with vehicle or 10 nM Ins in the presence or absence of 1 µM IR inhibitor OSI-906, 1 µM PI3K inhibitor alpelisib or 100 nM MEK inhibitor trametinib. C Protein levels of c-Fos in BCAHC-1 cells exposed for 6 h to vehicle or 10 nM Ins alone or in combination with 2 mM metfomin (Met), which was added to culture medium 18 h before the treatment with vehicle or insulin. D BCAHC-1 cells were transiently transfected for 18 h with c-Fos and then cells were treated for 12 h with vehicle or 10 nM Ins alone or in combination with 2 mM Met, which was added to culture medium 18 h before the treatment with vehicle or insulin. E Immunoblots of Cyclin D1 from BCAHC-1 cells treated for 6 h with vehicle or 10 nM Ins in the presence or absence of 1 µM IR inhibitor OSI-906, 1 µM PI3K inhibitor alpelisib or 100 nM MEK inhibitor trametinib. F BCAHC-1 cells were transiently transfected for 18 h with a Cyclin D1 promoter construct, then cells were treated for 12 h with vehicle or 10 nM Ins in the presence or absence of 1 µM IR inhibitor OSI-906, 1 µM PI3K inhibitor alpelisib or 100 nM MEK inhibitor trametinib. G Immunoblots of Cyclin D1 from BCAHC-1 cells transfected for 24 h with a vector or a dominant-negative c-Fos construct (DN/c-Fos) and then exposed for 6 h to vehicle or 10 nM Ins. H Protein levels of Cyclin D1 in BCAHC-1 cells exposed for 6 h to vehicle or 10 nM Ins alone or in combination with 2 mM Met, which was added to culture medium 18 h before the treatment with vehicle or insulin. Side panels show densitometric analysis of the blots normalized to β-actin. Results shown are representative of at least three independent experiments. I BCAHC-1 cells were transiently transfected for 18 h with a Cyclin D1 promoter construct, then cells were treated for 12 h with vehicle or 10 nM Ins alone or in combination with 2 mM Met, which was added to culture medium 18 h before the treatment with vehicle or insulin. The luciferase activities were normalized to the internal transfection control, and values of cells receiving vehicle were set as onefold induction on which the activity induced by treatments was calculated. Columns represent 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 Ins treated sample (p < 0.05)