Gene Expression Analysis of Cells Treated with Fruti

Human MCF-7, HepG2 and Fibroblast cells were seeded in 12 wells plate. After reaching confluence, cells were incubated with vehicle (0) or fruti at 10, 25, 50, 75 or 100 µM for 24 h (concentrations previously chosen by WST1 assay screening). Total RNA was extracted from cultured cells using Trizol reagent (Sigma-Aldrich, St. Louis, Missouri, USA) and measured by Nanodrop 100 (Thermo Scientific, Waltham, Massachusetts, USA). Complementary DNA was synthesized from 2 µg total RNA with an oligo-dT, random hexamers primers and deoxythymidine oligomer, Ribolock RNAse inhibitor and RevertAid reverse transcriptase. Real-time polymerase chain reaction was performed in a Lightcycler apparatus (Roche, Mannheim, Germany) using the sensiFAST SYBR No-ROX kit (Bioline, London, UK). Initial fluorescent values were calculated by LinRegPCR3 (version 2013.0; Academic Medical Center, Amsterdam, The Netherlands). Primer sequences were described in Supplementary Table S2. The most stable reference genes were calculated using geNorm^{40 (link)}. Transcript levels were normalized to housekeeping genes ratio GAPDH and Cyclophilin (CyP) for MCF-7 and Fibroblasts cells, and the genes ratio HPRT and Actin for HepG2 cells.