Cell culture and MTT assay

H295R cells were cultured as previously described [16]. Cell monolayers were subcultured onto 60 mm dishes for protein and RNA extraction (4 × 10⁶ cells/plate) and 12 well/plate (1 × 10⁵ cells per well) for the MTT assay. Prior to experiments, cells were starved overnight in DMEM/F-12 medium without phenol red. Cells were treated with (G-1, 1μM) (Tocris Bioscience, Bristol, UK) in DMEM/F-12 containing 2, 5% FBS-DCC (fetal bovine serum dextran-coated charcoal-treated). Inhibitors such as PD98059 (10μM), SB203580 (10μM), SP600125 (10 μM) (Calbiochem, Merck KGaA, Darmstadt, Germany) and ROS scavenge molecule NAC (N-acetyl cysteine, Sigma) (5mM) were used 1h prior to G-1. Cell viability was measured using MTT assay (Sigma-Aldrich) as already reported [43]. Each experiment was performed in triplicate and the optical density was measured at 570 nm in a spectrophotometer. Experiments were repeated three times.