Proliferation assay

Cell survival was determined using the Cell Counting Kit-8 (CCK-8) assay (Sigma, UK). U87MG cells were seeded at a density of 8,000 cells/well in 96-well plates and allowed to adhere overnight at 37°C in a humidified atmosphere of 95% air and 5% CO₂. Fucoxanthin and LY-294002 were tested in triplicate in at least two separate experiments. Dilutions of fucoxanthin and LY-294002 were made first at different concentrations in DMSO and then added to cell medium at a 1:100 ratio. Then, the culture medium was removed from the plates, and fresh medium containing compound was added. Control cells were treated with vehicle solution containing 1% DMSO. Blank controls without cells were also prepared. After 72h treatment, 5 μL of CCK-8 was added to every well containing 100 μL of tested compounds, controls or blank. After 3 h of incubation at 37°C in the dark, the plates were read using a Mithras LB940 multimode microplate reader (Berthold Technologies), and the absorbance values were determined at 490 nm, according to the manufacturer`s instructions. The percentage of surviving cells was calculated for each well using the formula:

where A_t is absorbance of the medium with tested compound, A_c is absorbance of control medium, and A_b is absorbance of blank medium.

Concentration-effect relationships for both compounds were analyzed using Prism software version 8.0.1 (GraphPad, Inc., San Diego, CA). Data were fitted using a four-parameter logistic equation.