Tc 100 medium

Bombyx mori ovary cell line BmN-SWU1 was established and preserved by our laboratory, and grown in TC-100 medium (United States Biological, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 100 U/ml penicillin and 100 μg/ml streptomycin42 (link). After the cells were grown to 80% confluency, the plasmids were transfected with the cells using the X-tremeGENE HP DNA Transfection Reagent (Roche, Switzerland). Briefly, 1 × 10⁵ cells were transfection with 0.8 μg of ATAD3A, HSPD1 and LEF-11 expression plasmids and at 6–8 hour post transfection, the medium was replaced with medium containing 10% FBS.

The B. mori ovary cell line BmN-SWU1 was cultured at 27 °C in TC-100 medium (United States Biological, Salem, MA, USA) supplemented with 10% (V/V) fetal bovine serum (Gibco, Gaithersburg, MD, USA) and 10% (V/V) penicillin/streptomycin [40 (link)]. Recombinant BmNPV (vA4^prm-EGFP) containing an EGFP marker gene driven by the B. mori actin A4 promoter was created from the bacmid bMON7214, which contains the BmNPV genome [41 (link), 42 (link)]. The BmN-SWU1 cells were transfected with the vA4^prm-EGFP construct, and viral titers were determined using the 50% tissue culture infective doses assay [42 (link)].

The Bombyx mori ovary cell line BmN-SWU1 (BmNS) was cultured at 27°C in TC-100 medium (United States Biological, Salem, MA, USA) supplemented with 10% (v/v) fetal bovine serum (Gibco, Gaithersburg, MD, USA) and 2% (v/v) penicillin/streptomycin (Gibco, Gaithersburg, MD, USA). Bacillus thuringiensis (BT) is maintained in our laboratory.

The Sf9 ovary cell line from Spodoptera frugiperda, the BmE embryonic cell line from B. mori, and the BmNS ovarian cell line from B. mori larvae were all maintained in our laboratory. The Sf9 and BmE cell lines were maintained in Grace’s medium (Gibco), and the BmNS cell line was maintained in Tc100 medium (US Biological) supplemented with 10% fetal bovine serum (Gibco).

The B. mori ovary cell line, BmN-SWU1, was established and preserved at our laboratory [36 (link)], and was cultured at 27 °C with TC-100 medium (United States Biological, Swampscott, MA, USA) supplemented with 10% fetal bovine serum (BIOAGRIO, Mountain View, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, Grand Island, NY, USA).
BmN-SWU1 cells were plated in cell culture plates (Corning Incorporated, Corning, NY, USA) and grown to 80% confluence. Then, the plasmids or bacmids were transfected into the cells using TransIT^®-Insect Transfection Reagent (Mirus, Madison, WI, USA) according to the manufacturer’s instructions.

The silkworm P50 strain was maintained in the Key Laboratory of Sericulture, Anhui Agricultural University, Hefei, China. The larvae were fed using fresh mulberry leaves. The first three instar larvae were reared at 26 ± 1 °C, 75 ± 5% relative humidity with 12 h day/night cycles, and the last two instar larvae were reared at 24 ± 1 °C, with the same relative humidity and photoperiod as above. The BmN cell line was cultured in TC-100 medium (United States Biological, Swampscott, MA, USA) supplemented with 10% (volume ratio, v/v) fetal bovine serum, 100 μg mL⁻¹ penicillin and 30 μg mL⁻¹ streptomycin (Gibco, Rockville, MD, USA) at 27 °C.
The BmNPV T3 strain was kept in our laboratory. On the 1st day of the 5th instar, all larvae were starved for 24 h, then administered 5 μL BmNPV suspended in water (1.0 × 10⁶ OBs mL⁻¹) per os, and the control was treated with 5 μL sterile water. Larvae midguts were collected at different time points post infection.
Budded virus of BmNPV containing an EGFP tag (BV-EGFP) was generously donated by Doctor Xue-Yang Wang from Jiangsu University of Science & Technology and was kept in our laboratory. The culture containing BV-EGFP (1.0 × 10⁸ pfu mL⁻¹) was used to infect BmN cells in this study.

The Bombyx mori cell line BmN, which was kindly gifted by Dr. Xudong Tang from Silkworm Pathology Laboratory in Jiangsu University of Science and Technology, was cultured at 27 °C in TC-100 medium (United States Biological, USA) supplemented with 10% (V/V) fetal bovine serum (FBS) (Gibco, USA) in 6-well plates to a confluency of about 70%. BmN cells per well were infected with recombinant BmNPV BVs containing an egfp marker gene at a MOI of 5. The P50 silkworm strain was reared at room temperature and under a photoperiod of 12 h light and 12 h dark until the fourth molt. For viral inoculation, 2 μL BmNPV viral stock including recombinant BmNPV BVs was injected into each larva through intersegmental membrane. The control uninfected larvae were injected with 2 μL 0.9% NaC1 solution. The infected group and the uninfected group had 3 replicates respectively and each replicate was pooled with 5 larvae.