Quantifying Proliferation in HCC Cells

HCC cell lines were cultured for 24 h in 1% FBS medium and seeded in 96 multi-well plates. After attachment of the cells to the dishes, in addition to the described treatments, IGF1 75 ng/mL and VEGF 20 ng/mL recombinant molecules were also used as single treatment. The proliferating cells were evaluated by the staining with anti-human Ki-67 (BioLegend Inc., San Diego, CA, USA) diluted 1:200 in PBS (Cell Signaling, Beverly, MA, USA) for 2h in a humidified dark chamber. The staining was performed as previously described [32 (link)]. The ImageJ software (http://rsb.info.nih.gov/ij/) was used for the analysis of fluorescent signals and the values relative to three independent experiments were plotted in graphs using GraphPad Prism 5.0 software (La Jolla, CA, USA).