Cck 8 solution

For the Cell Counting Kit-8 (CCK-8) assay, A2780 and SKOV3 cells were seeded in 96-well plates at a density of 3,000 cells/well. For verification of FOXM1 inhibitors regarding the proliferation of A2780 and SKOV3 cells, Cisplatin (100 μM) and FOXM1 inhibitor (100 μM) treated cells were cultured for 24 h. For the effects of DZY-4 on the proliferation of A2780 and SKOV3 cells, DZY-4 (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μM) or Cisplatin (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 μM) treated cells were cultured for 24 h. Subsequently, 100 μl of complete medium supplemented with 10 μl of CCK-8 solution (TOPSCIENCE, C0005) was added to each well, and the plates were incubated for 2 h. Finally, absorbance was measured at 450 nm.

A549 cells were plated into a 96-well plate and grown overnight, and then incubated with Bet v 1 or RSL-3 (HY-100218A, MCE, USA) at the indicated concentration in the presence or absence of 20 μM Fer-1 (HY-100579, MCE, USA), 20 μM DFO (HY-B0988, MCE, USA), 100 μM Z-VAD-FMK (HY-16658B, MCE, USA), 100 μM Nec-1 (T1847, Topscience, USA). Afterwards, cell counting kit-8 (CCK-8) solution (Cat #C0005, Topscience, China) was applied to each well, and the plate was subjected to one hour of incubation. The absorbance was determined at 450 nm.

The cell lines A549-mCALR, H460-mCALR, LLC-mCALR, and their controls (2000 cells) were seeded into 96-well plate with 100 μl RPMI-1640 medium (10% FBS). Each group set up blank control without cells in parallel with experimental group. The 96-well plates were incubated respectively at 37°C under 5% CO₂ for 0, 24, 48 or 72 hours. According to the manufacturer’s protocol, each well was added 10 μl CCK8 solution (C0005, Topscience) for 2 hours. The absorbance at 450 nm was measured using a microplate reader.