Cck 8

Human liver cancer cell lines HepG2, Bel-7404, and human umbilical vein endothelial cells (HUVECs) (ATCC, Manassas, VA, United States) were grown in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin antibiotic. Human renal proximal tubular epithelium HK-2 and human colorectal cancer cell line HT-29 (ATCC, Manassas, VA, United States) were grown in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin antibiotic. All the cells were maintained at 37°C with 5% CO₂ under a humidified atmosphere. For the cytotoxicity assay, cell viability was tested using the Cell Counting Kit-8 (CCK-8, DojinDo, Japan). The cells were seeded in 96-well plates with 5,000 cells (100 μL) per well, allowed to attach for 6–8 h, and then exposed to CD437 at different concentrations (100 μL), prepared in PBS or 0.1% DMSO (negative control), for 24 h. CCK-8 (10 μL) was then added to each well and the plates were incubated at 37°C for 1–4 h. The absorbance was measured at 450 nm, and the cytotoxicity was calculated using the following formula:
The 50% inhibitory concentration (IC₅₀) value was determined using GraphPad Prism 8 (GraphPad Software Inc., CA, United States). The assay was carried out in triplicate.

Cell counting kit-8 (CCK8) (Beyotime Biotechnology, Shanghai, China) was used for cytotoxicity analysis. Cells (5 × 10³/well) were plated in 96-well plates and stimulated with drugs at different concentration gradients for 24 h. CCK-8 solution was added and incubated in an incubator for 3 h. The absorbance value was measured at 450 nm, and Cancer Chemopreventative Properties (IC50) as calculated via GraphPad Pro Prism8.0 (GraphPad, San Diego, CA, USA). As for colony formation assay, cells (1 × 10³/well) were plated in 12-well plates and cultured for 24 h. Stimulation with different drug concentrations was added and cultured for 7 days. Cloned cell colonies were fixed, stained, and imaged.

Human NPC cell lines 5-8F and HNE3 were cultured in RPMI-1640 medium supplement with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO₂ atmosphere. Cell cytotoxicity and viability assays were performed by using CCK-8 (Dojindo) according to the manufacturer’s instructions. The chemicals 17-AAG, NMS-873, and Ro-3306 were purchased from Selleck (Selleck Chemicals) and dissolved in dimethyl sulfoxide (DMSO). For IC50 determination, 5-8F and HNE3 cells were seeded into 96-well plates at 2 × 10³ cells per well and treated with a series dilution of the three chemicals mentioned above for another 72 h. Cell viability was examined by a PowerWave XS Microplate Reader (BioTek) at 450 nm absorbance after incubation with CCK-8 for 1 h. The values were determined using GraphPad Prism 7.