Cck 8

The effect of ISM1 expression on human colon cancer cell lines was analyzed using the CCK8 (Meilunbio, Shanghai, China) assay. A 200 μL cell suspension containing 5,000 cells was seeded in each well (96-well plate). At different time points (0, 24, 48, and 72 h), the supernatants were removed, and 100 μL CCK8 solution (10 μL CCK8: 90 μL medium) was added to each well and incubated with the cells for 3 h before analysis. The absorbance at 450 nm was measured.

A total of 5000 cells were plated into a 96-well plate and incubated for 24 h. The cells were then exposed to drugs for 24 h. Subsequently, 100 μL of fresh medium containing 10% cell counting Kit-8 (CCK8) solution (MA0218, Meilunbio, Dalian, China) was added. The number of cells was determined by measuring the absorbance at 450 nm using a 96-well plate reader (PerkinElmer, Hopkinton, MA, USA). For the colony formation assay, 500 cells were seeded into each well of a 6-well plate in triplicates under different conditions and incubated for 14 days. Colonies were fixed with paraformaldehyde and stained with crystal violet. Average colony counts were calculated, and a paired t-test was used to evaluate statistical significance.

CCK-8 (Meilun Biotechnology, Dalian, China) was used to detect the proliferation of CRC cells. During the experiment, cells were seeded in a 96-well plate at a density of 2 × 10³ cells per well. After attachment, cells were transfected with siRNA and incubated in an incubator at 37 °C with 5% CO₂. At 24, 48, 72, and 96 hours after transfection, medium containing 10% CCK-8 was placed into each well, and the cells were incubated at 37 °C for 30 min. Then, the absorbance was detected at a wavelength of 450 nm with a microplate reader (Tecan, Lyon, France).

HaCat cells were seeded at a density of 3 × 10⁴ ml⁻¹ in 96-well plates. After 24 h, HaCat cells were treated with 800 μM hydrogen peroxide (H₂O₂) to mimic extracellular high ROS environment and co-treated with low (50 μg/mL, L-FE), medium (200 μg/mL, M-FE), and high (500 μg/mL, H-FE) concentrations of FE. Subsequently, cell proliferation rates were determined using the cell counting kit-8 (CCK-8, Dalian Meilun Biotechnology Co., Ltd., Meilunbio), and living and dead cells were identified using Calcein/Propidium Iodide (PI) Cell Viability/Cytotoxicity Assay Kit (Beyotime, Shanghai, China). A fluorescence microscope (EVOS M700, Thermo Fisher, United States) was used to calculate cell viability with the following formula: $\text{Cell\hspace{0.17em}viability\hspace{0.17em}}\left(\%\right)=\frac{\text{Number}\text{of}\text{Calcein}+\text{Cells}}{\text{Number}\text{of}\text{Calcein}+\text{Cells}+\text{Number}\text{of}\text{PI}+\text{Cells}}\times 100$