Effects of conditioned media (CM) on viability of cancerous and non-cancerous cells. (A) Hep3B cells were seeded in a 96-well plate at a density of 5 × 103 and 1 × 103 cells/well in RPMI-DMEM/F12 medium conditioned by Spalax or mouse skin newborn fibroblasts (SpNbF and MNbF, respectively). Hep3B cells were incubated for four days; viability was estimated by PrestoBlue® Reagent. (B,C) Hep3B cells (1 × 104 cell/well) were cultured in six-well plates under conditioned medium of Spalax adult skin fibroblasts (B) or grown in medium generated by Hep3B cells (C). After nine days, the cells’ survival rates were assessed by a Countess® cell counter (Life Technologies); red: dead cells, blue: viable cells. (D) Hep3B and HepG2 cells were incubated under Spalax CM for four days, followed by changing the media either to fresh media or new Spalax CM. After two days, viability was estimated by PrestoBlue® Reagent. (E) Spalax fibrosarcoma cells (SpFS2240) were incubated for three or seven days in full medium or under CM of Spalax adult skin normal fibroblasts (SpAdF CM), Hep3B (Hep3B CM), Spalax fibrosarcoma (SpFS2240 CM). Cell viability was evaluated by using PrestoBlue® reagent. Results are presented as percentage of control (SpFS2240 CM); mean ± S.D. (F) Effects of CM generated by Spalax or mouse normal fibroblasts (SpNbF CM and MNbF CM, respectively) on the growth of non-cancerous cells. The viability was estimated after four days by PrestoBlue® reagent; mean ± S.D. (G) Heat treatment of conditioned media. Seven-day CM, generated by Spalax or rat fibroblasts, was heat-treated at 56°C for 10 minutes and 30 minutes prior to addition to Hep3B cancer cells (2,000 cell/well) in 96-well plates. Cells were incubated for seven days followed by PrestoBlue® test. All results were obtained from three independent experiments performed in three to six technical repeats.