Sf9 cells

Spodoptera exigua (Hübner) were purchased from Henan Jiyuan Baiyun Industry Co., Ltd. Sf9 cells (S. frugiperda ovary cells), Thiazole blue (MTT) and Dimethyl sulphoxide (DMSO) were purchased from Wuhan Procell Life Technology Co., Ltd. One Step TUNEL Apoptosis Assay Kit and universal genomic DNA purification mini spin kit were purchased from Shanghai Beyotime Institute of Biotechnology.

Sf9 cells (Procell, Wuhan, China) were cultured in insect-cell medium (Sino Biological, Beijing, China) in a cell-culture flask at 27 °C. When the cell confluence reached 80–90%, cells were resuspended in insect-cell medium, and the cell density was adjusted to 2 × 10⁵ cells/ml. Sf9 cells (100 μl) were added to each well of a 96-well plate (Corning, Guangzhou, China) and incubated at 27 °C for 24 h. Different final concentrations of rHtA (0, 1, 2, 4, 8, 16, 32, and 64 ng/ml) were added and incubated at 27 °C for 48 h, and cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 10 μl MTT solution (5 mg/ml MTT in PBS) was added to each well of the 96-well plate, followed by incubation at 27 °C for 4 h, after which media supernatants were discarded carefully. Dimethyl sulfoxide (100 μl) was then added to each well to dissolve the formazan for 10 min at 37 °C. Absorbance was measured on an ELISA plate reader (BioTek ELX800, BioTek Instruments Inc., Vermont, USA) at a wavelength of 570 nm.