Cytochalasin D Cytotoxicity Evaluation

Cytochalasin D (Cyt D) (Sigma-Aldrich, St. Louis, MO, USA) was used as the inhibitor of actin polymerization. A CCK-8 assay was performed to evaluate the cytotoxicity of Cyt D to EBL cells (Dojindo, Shanghai, China), as previously described [11 (link)]. Briefly, 5 × 10³ cells/well were seeded and allowed to grow for 24 h in 96-well plates and then treated with Cyt D (0.1 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, 10 μM, or 20 μM) for 24 h. Cells treated with DMSO were used as the negative control, and cells with no treatment were used as the mock group. Next, each well was incubated with 10 μL CCK-8 for 2 h. The light absorption value at 450 nm was detected by the microplate spectrophotometer (PerkinElmer Victor NIVO 3S, Waltham, MA, USA). Each treatment was carried out in three repeats, and all experiments were performed independently three times. After that, the monolayer EBL cells (2 × 10⁵ cells in a 12-well plate) were treated with the selected concentration of Cyt D for 2 h, followed by the addition of 8 μg/mL rMbovP0145 and incubation for more than 12 h. The total RNA was then extracted, and qRT-PCR was used to analyze the expression of IL-8 mRNA.