*4.3. Cell Proliferation Assay*

The cell proliferation was determined using the MTT test (Sigma Aldrich, Steinheim, Germany). In brief, 1 <sup>×</sup> 104 (NIH/3T3 and J77.4.E) or 1 <sup>×</sup> 105 (canine cancer cell lines) cells per well were seeded in a 96-well-plate (Thermo Fisher Scientific, Denmark), and the tested chalcones were added in the increasing concentrations (1.5, 3.125, 6.25, 12.5, 25 and 50 μM). After incubation for 48 h, 20 μL of MTT solution (5 mg/mL) were added to each well. After the contents dissolved, the optical density of wells was measured with a microplate reader (Spark, Tecan) at a reference wavelength of 570 nm. The values were means from three independent experiments (three wells each).
