Tc 100

Silkworm strains, YeA, YeB and p50 (Dazao), were maintained in the Key Laboratory of Sericulture, College of Life Sciences, Jiangsu University of Science and Technology, Zhenjiang, China. The first three instars of larvae were fed fresh mulberry leaves at 26 ± 1 °C and relative humidity of 75 ± 5% on a 12-h daily cycle, whereas the final two instars were fed at 24 ± 1 °C, and other conditions remained unchanged.
A BmN cell line derived from the silkworm ovary was cultured in TC-100 (AppliChem, Darmstade, Germany) medium supplemented with 10% (v/v) fetal bovine serum (FBS) (Thermo Fisher Scientific, New York, USA) and 1% (v/v) penicillin and streptomycin (AppliChem, Darmstade, Germany) at 28 °C.

Bm5 cells (Bombyx mori-derived cell line) were cultured and maintained at 27 °C with 10% fetal bovine serum (FBS, Gibco, USA) in TC100 (insect cell culture medium) (Applichem, Darmstadt, Germany) as per the published literature [18 ]. For co-transfection, Bm5 cells were cultured at a constant density of 1 × 10⁶ cells per well in six well plates for 12 hours with TC100 media containing FBS. TC100 media without FBS was used to wash the cells twice and a mixture of transfection and co-transfection was introduced to cells. Between 4–6 h post-transfection, FBS was introduced to the cell culture media. For viral amplification and expression, cells were infected with a multiplicity of infection (MOI) of 0.1 for 1–2 h.

The pFastBac dual vector, pGL-A3-luc-sv40 vector and BmBacJS13 were provided by the Key Laboratory of Sericultural Research Institute, Chinese Academy of Agricultural Sciences (CAAS). Primer synthesis and sequencing were performed by Sangon Biotech (China). RIPA Lysis Buffer (Strong), SDS–PAGE Gel Kit (CWBIO), One Step Western Kit HRP (Mouse) and TC-100 were purchased from Applichem (Germany), and foetal bovine serum was purchased from Corning (USA). The plasmid extraction kit, RT-PCR kit and SYBR Green PCR kit were obtained from Vazyme Company.

BmN cells were plated in 12-well tissue culture plates (2 × 10⁵ cells/well; Asahi glass, Yokohama, Japan) and maintained in normal growth medium (TC-100 [Applichem, Darmstadt, Germany], 10% FBS, and 50 μg/mL gentamycin). At 3 days post-seeding, the cells were pre-incubated in 800 μL of assay medium (TC-100 supplemented with 1 mM 1-methyl-3-isobutylxanthine [IBMX; Sigma-Aldrich, Tokyo, Japan]) and 1 mM phenylmethylsulfonyl fluoride for 10 min at 28°C. Following pre-incubation, 100 μL of a potential competitor and 100 μL of the peptide of interest were added sequentially to the medium and incubated at 28°C for 30 min. After incubation, the cells were washed twice with TC-100 and then immediately frozen in liquid nitrogen. Intracellular cAMP and cGMP were extracted by sonication with 300 μL 0.1 M HCl supplemented with 1 mM IBMX and then collected from the supernatant subsequent to centrifugation (4°C, 16,000 × g, 10 min). The extracted cAMP and cGMP were quantified in duplicate using a cAMP ELISA kit (Cayman Chemicals, Ann Arbor, MI, USA) and a cGMP EIA system (GE Healthcare, Hino, Japan), respectively. These experiments were repeated at least twice.

The BmN cells derived from the silkworm ovary were cultured in TC-100 (AppliChem, Gatersleben, Germany) that contained 10% (v/v) fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% double-antibiotics (penicillin and streptomycin) at 28 °C [24 (link)]. The siRNA and overexpression vector of Bmapaf-1 were transfected into BmN cells using the Neofect ^TM DNA transfection reagent (NEOFECT, Beijing, China). Briefly, BmN cells were seeded into 30 mm culture flasks (approximately 1 × 10⁵ cells/well) before transfection. Then, 4.0 µg of siRNA or vector and 4.0 µL of transfection reagent were added successively into 200 µL of serum-free TC-100 to prepare the transfection solution, which was added into the culture medium to finish the transfection. The best efficiency was obtained at 24 h, and this time point was selected for all subsequent analysis.
The fluorescence signal in this study was captured at 24, 48, 72 h post-inoculation after knockdown or overexpression of Bmapaf-1 at 24 h using an inverted microscope DMi3000B camera (Leica, Solms, Germany), and the picture was processed with the Application Suite V4.6 software (Leica, Germany).