Bl21 de3 cells

Goldfish MCSF-2 ORF excluding singnal peptide was amplified by PCR using gene-specific primers that included 5’-end BamH I and Hind III insertions (Table S1). Restriction enzymes BamH I and Hind III were used to thoroughly digest the PCR result. (Thermo Fisher Scientific, USA), then ligated to the pET32a (+) vector digested with BamH I/Hind III. This resulted in the creation of the recombinant plasmid pET32a-gfMCSF-2, which were subsequently converted into competent E. coli (BL21/DE3) cells (TransGen, China). To express the RgMCSF-2 proteins, 0.1 mM IPTG induction was carried out at 16°C overnight, and recombinants proteins expression was analyzed by SDS-PAGE. Purification of the recombinant protein was accomplished by using Ni-NTA Sefinose Resin (Sangon, China) as directed by the manufacturer. As a control protein in subsequent experiments, the pET32a (+) vector containing a thioredoxin tag without an insert was expressed and purified in the same manner. Subsequently, a ProteoSpin Endotoxin removal column (Norgen Biotek, USA) was used to purify the recombinant protein after it was dialyzed overnight at 4°C in 1 x PBS. Micro BCA Protein Assay Kit (Beyotime, China) was used to determine protein content.

B. mori strain p50 was used in this study. G. pyloalis was collected in the mulberry field of Anhui Agricultural University. Larvae were reared on fresh mulberry leaves at 25–26 °C under a 12:12 h (L:D) photoperiod with 70% relative humidity. The silkworm ovary-derived BmN cells were routinely maintained at 27 °C in TC100 medium (Ximeijie, China) supplemented with 10% fetal bovine serum (ExCell, Shanghai, China) and 1% penicillin-streptomycin (Solarbio, Beijing, China) in our laboratory. The expression vector pET-24b was purchased from Invitrogen. The pET24b-BmSuc1 recombinant plasmid and the pFastBac-dual vector are always reserved in our laboratory. The E. coli strain BL21 (DE3) cells and DH10Bac (BmNPV) cells were purchased from Transgene Biotech (Beijing, China).

Escherichia. coli strain BL21(DE3) cells (used for gene expression) and TOP10 cells (used for molecular cloning) were purchased from TransGen Ltd. (China) The gene P450_BsβHI (derived from P450_Bsβ (NCBI Reference Sequence: WP_119898938.1) with a Q85H/V170I mutagenesis) was synthesized by Inovogen Ltd. (China). Fast Mutagenesis kit was from Vazyme Biotech Co., Ltd (China). Plasmid extraction kits and gel extraction kits were obtained from Omega Bio-tek (USA). Capric acid, lauric acid, myristic acid, stearic acid, undecene and tridecene were purchased from BioRo Yee Ltd. (China). All chemicals used were of analytical grade.

The GST-IDO1 gene was cloned by Shanghai Generay Biotech Co. Ltd (Shanghai, China). The BL21(DE3) cells were obtained from TransGen Biotech Co. Ltd (Shanghai, China). Ampicillin, isopropyl β-D-thiogalactoside (IPTG), 5-Aminolevulinic acid (5-ALA), phenylmethanesulfonyl fluoride (PMSF), DNase I (from bovine pancreas), reduced glutathione, L-tryptophan, ascorbic acid, methylene blue, catalase, and sodium dithionite were obtained from Sangon Biotech (Shanghai, China) Co. Ltd. DMSO was purchased from Sigma-Aldrich. The CCK8 kit was purchased from Beyotime Biotechnology Co. Ltd.(Shanghai, China). Glutathione agarose beads were purchased from Changzhou Smart-Lifesciences Biotechnology Co. Ltd. (Changzhou, China) PD-10 desalting column was purchased from GE Healthcare Biosciences. HRV 3C Protease was purchased from Sino Biological Inc. (Shanghai, China). Cancer cell lines were purchased from the Cell Bank of Chinese Academy of Sciences. The experimental compounds were provided by Professor Zhao (Shanghai Institute of Organic Sciences, Chinese Academy of Sciences). All chemicals of analytical and reagent grade were obtained from commercial sources and used without further purification.