Fetal bovine serum (fbs)

Quiescent satellite cells were collected from the skeletal muscles of 8-week-old male SJS-model mice using fluorescence-activated cell sorting and an SM/C-2.6 antibody, as described previously [31 (link)]. The SM/C-2.6 antibody was provided by Dr. Fukada. These satellite cells were seeded on rhLaminin-521 (Thermo Fisher Scientific)-coated plates and cultured in satellite cell growth media (Dulbecco’s modified Eagle’s medium (DMEM), 20% fetal bovine serum, 10% horse serum, chicken embryo extract (United States Biological, Salem, MA, USA), and penicillin/streptomycin) containing 2.5 µg/mL recombinant human FGF (ProteinTech, Rosemont, IL, USA), as previously described [32 (link),33 (link)]. Satellite cells were differentiated to myotubes by culturing in high glucose DMEM (Thermo Fisher Scientific), supplemented with 2% horse serum (Thermo Fisher Scientific) and penicillin/streptomycin. To rescue perlecan-null myotubes, recombinant perlecan protein (20 μg/mL; purified and provided by Dr. Sasaki) was added at the start of the differentiation process. Calcium imaging experiments were performed 12–16 h after adding 70 µg/mL laminin/entactin complex (Corning Inc., Corning, NY, USA) and 10 ng/mL recombinant neural agrin (R&D Systems, Minneapolis, MN, USA).

DaZao, a bivoltine strain of the silkworm, was obtained from the Silkworm Gene Bank of Southwest University, Chongqing, China. The non-diapause eggs were divided into two groups; one group was incubated at 25 °C (diapause induction temperature), while the other group was incubated at 15 °C (non-diapause induction temperature). All the eggs were incubated at the same light condition (continuous darkness). Four groups were prepared from embryos exposed to the two temperature treatments (25 and 15 °C) and in the two phases (the blastokinesis (BK) and head pigmentation (HP) phases). Due to the non-uniform development of embryos, we dissected a great deal of the silkworm eggs and selected the embryos showing the same morphology at the same developmental stage for subsequent sequencing. The resulting four groups were labeled BK25, BK15, HP25, and HP15. Each group comprised 100 embryos. All samples were stored at −80 °C for further use. The cell line, BmE, derived from the silkworm embryo, was cultured at 27 °C in Grace insect medium (Life Technologies, Shanghai, China) supplemented with 10% fetal bovine serum (United States Biological, Swampscott, MA, USA), penicillin (200 U/mL), and streptomycin (200 U/mL).