2.5 |. Insect cell culture, apoptosis phenotypes, and gene knockdown study

The L. decemlineata cell line (Lepd‐SL1 cells) and T. castaneum cells, BCIRL‐TcA‐CLG1 were received from Dr. Cynthia Goodman from Biological Control of Insecťs Research Laboratory (USDA‐ARS, Columbia, MO) and cultured, as described previously (George, Gaddelapati, & Palli, 2019; Roy & Palli, 2018; Shukla et al., 2016; Yoon et al., 2016). The A. aegypti, Aag‐2 cells (Cui, Sui, Xu, Zhu, & Palli, 2014) were cultured in Schneider's insect medium with L‐glutamine (Sigma‐Aldrich, St. Louis, MO) containing 10% fetal bovine serum (Seradigm FBS, VWR^® Life Science). Sf9 cells were derived from IPLB‐Sf21‐AE, an established cell line originally isolated from S. frugiperda ovaries. The plasmids containing the genes, Luciferase and SID‐1 (dsRNA transporter channel) from C. elegans (Xu et al., 2013) were stably expressed in Sf9 cells and named Sf9_LUC_CeSID‐1 and maintained in Sf‐900^™ II SFM (Gibco^™, Thermo Fisher Scientific). All the cell lines were maintained in a 27–28°C incubator. For apoptosis phenotypes and gene knockdown studies, cells were seeded in 96‐ and 24‐well plates in 100 and 250 μl culture medium, respectively and exposed to dsGFP or dsmalE as control and dsIAP targeting the iap genes in nine insect species.