Escort 4

Hi5 and Sf21 tissue culture cells were maintained in IPL-41 medium containing 10% fetal calf serum as previously described (24 (link)). Recombinant bacmid DNA was isolated from DH10B host bacteria by the lysozyme method and 0.5-2.5 μg of total DNA was used to transfect Hi5 host cells using Escort IV (Sigma) according to established methods (29 (link)). Supernatants from transfected cells were collected 7-10 days after transfection and used to re-infect fresh host cells (30 ). Supernatants of infected cell cultures, obtained 7 days after infection, were stored at -70°C as high titer (> 10⁹ pfu/ml) stocks of recombinant infectious baculovirus particles.

NRCs were prepared according to [31 (link)], and C2C12 and HEK293 cells were prepared following standard protocols. NRCs, C2C12 and HEK293 cells were transfected using Escort III (Sigma), Lipofectamine 3000 (Invitrogen) and Escort IV (Sigma), respectively, according to manufacturer’s instructions. Following incubation at 37 °C, 5% CO₂ for 24–48 h for NRCs and HEK293 cells and up to 14 days for C2C12 myoblasts to allow differentiation into myotubes, cells were prepared according to [12 (link)]. Cells were stained with primary antibodies against myosin heavy chain (clone A4.1025) [9 (link)], obscurin domain O59 [61 (link)], titin Z1Z2 [15 (link)], p62/SQSTM1 (Abcam, ab41116161) and ubiquitin (Merck, FK2, 04–263) followed by Cy3 or Cy5-conjugated goat anti-rabbit or anti-mouse IgG (H + L) secondary antibodies (Jackson ImmunoResearch ML 115–165-146, 115–175-146, 111–175-144 and BioRad STAR36D549GA) and imaged on an LSM510 (Zeiss) or SP5 (Leica) confocal microscope. Antibodies were diluted 1:100 prior to incubation.

Exocyst subunits and PIP5K1C were expressed in SF9 cells cultured in Insect-XPRESS (Lonza) by baculovirus mediated expression. Baculovirus was produced using the FlexiBac methods.⁴⁴ (link) SF9 cells at 1x10⁶ cells/ml were co-transfected by the linearised Defbac viral backbone and the plasmids encoding for exocyst subunits or PIP5K1C using ESCORT-IV (Sigma). After 5 days, cells were checked for signs of viral transfection and 50-200 μl of supernatant of this P1 virus were used to infect 50 ml of insect cells to generate P2 virus. 5 days after infection, this P2 virus was again evaluated for virus production (LUNA-II; Logos Bio).

S2 cells were transiently transfected with the engineered plasmids using Escort IV (Sigma-Aldrich), according to the manufacturer’s instructions. Briefly, each well of a 6-well plate was filled with 3 × 10⁶ S2 cells and washed twice with serum-free culture medium. 14 μl of the Escort IV was first pre-incubated alone for 30–45 min in serum-free culture medium and then 30 min together with 1.5 μg of each of the pMT-FHV RNA1 plasmids plus 1.5 μg of the pMT-FHV RNA2 plasmid (1:1 ratio). The transfection medium was added to the cells and incubated for 16 h at 27°C. After incubation, the transfection medium was replaced with fresh medium containing serum (10% FBS) and further incubated for 24 h for cell recovery. Following cell recovery, copper sulfate (CuSO₄) was added to the medium to a final concentration of 700 μM to activate the plasmid promoter to express FHV RNA1 and 2. S2 cells were then observed for eGFP expression at 72 h post-activation using a Nikon Eclipse TS-100 microscope (Melville, NY, United States) and NIS Elements BR 4.11.00 imaging software (Nikon, Melville, NY, United States). The plasmid containing only eGFP (pMT-eGFP) was used as positive control for transfection and plasmid activation for eGFP expression, while the plasmid containing the wild type (WT) genome, pMT-FHV RNA1, was used as a negative control (no eGFP expression).

The Bm5 and High Five cell lines were maintained in a complete medium consisting of IPL-41 Insect Medium (Sigma-Aldrich), supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 0.25 μg/ml of amphotericin B (Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, Life Technologies). The cells were subcultured weekly and maintained at 27.5 °C.
Bm5 and High Five cells were transfected using Escort IV (Sigma-Aldrich), according to manufacturers’ instructions. An optimized volume of the transfection reagent was used, namely 3.7 μl and 15 μl per well of a 24- and 6-well plate, respectively.
In the overexpression experiments, the expression constructs were transfected overnight, at a concentration of 1 μg/ml. In addition, the pBmIE1 helper plasmid encoding the ie-1 gene for B. mori nuclear polyhedrosis virus^{48 (link)} was used, at a concentration of 0.3 μg/ml. In the knockdown experiments, the dsRNA was transfected at a concentration of 2.5 μg/ml. After the transfections, the medium was replaced for the complete medium above mentioned.