Grace medium

T. niembryonic cell line BTI-Tn5B1-4 (High Five) (
Granados et al. 1994
) and
S. frugiperdaovarian cell line Sf-9 (
Pasumarthy and Murhammer 1994 (link)
) were provided by Dr. Blissard, Boyce Thompson Institute of Cornell University.
T. niembryonic suspension cell line QB-Tn9-4s was established and preserved in our laboratory (
Meng et al. 2008
).
Autographa californicamultiple nucleopolyhedrovirus (AcMNPV-1A) (
Wood 1980 (link)
) and its β-galactosidase expressing recombinant strain AcMNPV-β-gal (
Wickham et al. 1992 (link)
) and secreted alkaline phosphatase (SEAP) expressing recombinant strain Ac-MNPV-SEAP (
Davis et al. 1992
) were kindly provided by Dr. Granados of Cornell University. All of the viruses were amplified and titrated following the plague assay method described by
Wood (1977)
using Sf-9 cells.
TNM-FH insect medium was prepared by supplementing Grace medium (Invitrogen,
www.lifetechnologies.com) with 0.3% lac-talbumin hydrolyzate (BD,
www.bd.com), 0.3% yeast extract (BD) and 10% fetal calf serum (FBS) (Thermo Scientific,
www.thermoscientific.com). Serumfree Sf-900 III medium was from Invitrogen and serumfree EX-CELL 420 medium was from SAFC Biosciences (Sigma-Aldrich,
www.sigmaaldrich.com). O-nitrophenyl-β-D-galactopyranoside (ONPG) and p-nitrophenyl phosphate (PNPP) were from Sigma-Aldrich.

Production of MCPyV and TSPyV VLPs in insect cells has been described previously [15] (link), [28] (link). VLPs were also generated for PtvPyV1, PtvPyV2 and OraPyV1. Briefly, VP1 coding sequences were obtained by total synthesis with codon usage-adapted sequences for expression in Spodoptera frugiperda cells (Genscript, Piscataway, NJ, USA) (Sequences based on PtvPyV1b (HQ385747), PtvPyV2c (HQ385750) and OraPyV1 (FN356900)) [26] (link), [27] (link). The different VP1 genes were cloned under the control of the polyhedrin promoter of pFastBac Dual plasmid (Invitrogen, FisherScientific, Illkirch, France) and then used to generate recombinant baculoviruses using the Bac-to-Bac system (Invitrogen). HiFive cells maintained in Grace medium (Invitrogen) were infected with the different recombinant baculoviruses for production of the polyomavirus VLPs. VLPs were then purified by ultracentrifugation (18 h at 30,000 rpm in a Beckman SW 32 rotor) in a CsCl gradient and assembly of VP1 into VLPs was verified by electron microscopy. The preparations were applied to carbon grids, negatively stained with 1.5% uranyl acetate and observed with a JEOL 1011 electron microscope at 50,000 nominal magnification (Figure 2).

Bm12 cells at logarithmic growth phase were used for transfection. Plasmid DNAs were mixed with Lipfectamine 2000 (Invitrogen) and added to cells in each well of 12-well culture plates with Grace medium (Invitrogen). To normalize the firefly luciferase activity, the renilla luciferase vector, pRL-SV40, was co-transfected with each of the pGL3-drived vectors containing tested promoters. After 6-h post transfection, the old medium was replaced with fresh Grace medium containing 10% FBS. The cells were cultured for additional 48 h at 28 °C before promoter activity assay. The cells were washed once with filtered PBS and then lysed in 200 μL Passive Lysis Buffer (Promega). Luciferase activity of the supernatant was analyzed using the Dual-Luciferase Assay System (Promega) according to the manufacturer’s instruction with a luminometer (IBA7300, Veritas, Turner Biosystems). All assays were conducted three times.

Bombyx mori strain Dazao was obtained from the Research and Development Center of the Sericultural Research Institute of the Academy of Agricultural Sciences of Guangdong Province, China. Larvae were reared on fresh mulberry leaves at 25 °C and a photoperiod of 12 h light:12 h darkness.
For methylation inhibitor treatments, 5-aza-dC (Sigma) was dissolved in 1X phosphate buffered saline (PBS). Two microliters of 5-aza-dC at the concentration of 10 μg/μL was injected into the hemolymph in the thoracic region of larvae at the second day of wandering stage (W2), the wings of 2 or 3 day-old pupa (PD2 and PD3) after 5-aza-dC injection were collected for RNA isolation. The same volume of 1× phosphate buffer saline (PBS) was injected as control. All data included three biological replicates, each with three technical repeats.
A B. mori cell line DZNU-Bm-12 (Bm12) originally developed from ovarian tissues [49 (link)] was maintained at 28 °C in Grace medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Hyclone).

Bombyx mori larvae strain P50, obtained from the Research and Development Center of the Sericulture Research Institute of the Academy of Agricultural Sciences of Guangdong Province at China, were reared on fresh mulberry leaves at 27 °C with a 12 h light/12 h dark cycle. Under this condition for 6 days, the fifth instar silkworm larvae started wandering, followed by the beginning of larval–pupal transition. After pupation, the newly molted pupae (P0 stage) were transferred to a clean and dry paper box and reared at 27 °C for 7 days before enclosing into adult moth.
Bombyx mori Bm12 (DZNU-Bm-12) cell line, originally derived from the ovarian tissues [50 (link)], was cultured in Grace medium (Invitrogen, California, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Utah, USA) at 28 °C.

Bm12 cells at their logarithmic growth phase were inoculated in culture media in 12- or 24-well culture plates (Corning, New York, NY, USA) and cultured for 12 h. Cell transfection and co-transfection were conducted when the cells were at approximately 80% density. Plasmid DNAs were mixed with Fugene HD transfection reagent (Promega, Madison, USA) and added to cells in each well of 12- or 24-well culture plates with Grace medium (Invitrogen). To normalize the firefly luciferase activity, the renilla luciferase vector, pRL-SV40, was co-transfected with each of the pGL3-derived vectors containing tested promoters. The cells were cultured for additional 48 h at 28 °C, followed by the luciferase activity assay, protein or RNA isolation.
For luciferase activity measurement, the cells were washed twice with filtered PBS and then lysed in 100 μL Passive Lysis Buffer (Promega, Madison, WI, USA). The samples were centrifuged at 800g for 5 min at room temperature. The supernatant was used to analyze the luciferase activity using the Dual- Luciferase Assay System according to the manufacturer’s protocol with a luminometer (IBA7300, Veritas, Turner Biosystems). The luciferase activity was normalized to the renilla luciferase activity. All assays included three biological replications and three technical replicates. The luciferase activity was represented as mean ± standard error (SE).