Lipofectin transfection reagent

H-2K^b-tsA58 (H2K) mdx mouse myoblasts were cultured and differentiated as described previously^{41 (link),45 (link),46 (link)}. Briefly, when 60–80% confluent, myoblast cultures were treated with trypsin (ThermoFisher Scientific; cat#: 15400054) and seeded on a 24-well plate at a density of 2 × 10⁴ cells/well. The plates were pre-treated with 50 μg/mL poly-D-lysine (Merck Millipore; cat#: P7886-50mg), followed by 100 μg/mL Matrigel (Corning Life Science; cat#: FAL354234). Myoblasts were differentiated into myotubes in Dulbecco’s Modified Eagle Medium (DMEM) (ThermoFisher Scientific; cat#: 11885084) containing 5% horse serum (ThermoFisher Scientific; cat#: 16050122) by incubating at 37 °C, 5% CO₂ for 24 h. AOs were complexed with Lipofectin transfection reagent (ThermoFisher Scientific; cat#: 18292011) at the ratio of 2:1 (v:w) (Lipofectin:AO) and used in a final transfection volume of 500 μL/well in a 24-well plate as per the manufacturer’s instructions.

The production of recombinant BivCaE proteins was performed using a baculovirus expression system [31 (link)]. The BivCaE cDNA, including His-tag sequence, was PCR-amplified using the following primer set: forward (1–18) 5′-ATGGAACTATCAGTTATC-3′ and reverse (1651–1668) 5′-TTACTCCTGCCCACTTAT-3′. The BivCaE cDNA was introduced into the baculovirus vector pBacPAK8 (Clontech, Palo Alto, CA, USA) to construct the pBacPAK8-BivCaE. For generating BivCaE protein-expressing recombinant baculoviruses, pBacPAK8-BivCaE (500 ng) was co-transfected with baculoviral DNA (100 ng) into insect Sf9 cells (1.0–1.5 × 10⁶ cells/well of a 6-well plate) using Lipofectin transfection reagent (Gibco BRL, Gaithersburg, MD, USA). Recombinant BivCaE proteins in insect Sf9 cells were produced by infection of recombinant baculoviruses [30 (link)]. Recombinant BivCaE proteins were purified using the MagneHis^TM Protein Purification System (Promega). Concentrations of recombinant BivCaE proteins were analyzed using a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA).

Recombinant catalase—GTPX, Tf, and SOD1—were produced using a baculovirus expression system [30 (link)]. The protein-coding sequences of catalase, GTPX, Tf, and SOD1 were PCR-amplified, and a His-tag sequence was included in each cDNA sequence. The cDNA fragments corresponding to the protein sequences were inserted into the pBacPAK8 transfer vector (Clontech, Palo Alto, CA, USA), in which gene expression is under the control of the polyhedrin promoter of Autographa californica nucleopolyhedrovirus (AcNPV). For the expression experiments, 500 ng of the constructs and 100 ng of AcNPV viral DNA [30 (link)] were co-transfected into 1.0–1.5 × 10⁶ Spodoptera frugiperda (Sf9) cells for 5 h by using Lipofectin transfection reagent (Gibco BRL, Gaithersburg, MD, USA). The transfected cells were cultured in TC100 medium (Gibco BRL) with 10% fetal bovine serum (FBS, Gibco BRL) at 27 °C for 5 d. Recombinant baculoviruses were propagated in Sf9 cells cultured in TC100 medium at 27 °C to produce the recombinant proteins, which were then purified using the MagneHis Protein Purification System (Promega, Madison, WI, USA). Protein concentrations were determined using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). The recombinant MRJP proteins (MRJPs 1–9) produced in baculovirus-infected insect cells were used as described in our previous studies [27 (link),28 (link)].

Sf21 cells were transfected with the respective expression plasmids at a density of 0.4–0.6x10⁶ cells/mL using Lipofectin Transfection Reagent (Life Technologies). A DNA concentration of 2 μg per 1x10⁶ cells was used at a Lipofectin: DNA ratio of 2:1. DNA and Lipofectin were incubated with the respective culture medium for precomplexing in 2.5% (v/v) of the final volume. After 30–60 min of incubation at RT the transfection mixture was diluted with medium in 37.5% (v/v) of the final volume and added to the cells.