In fect in vitro transfection reagent

MCF-7 (ATCC^® HTB-22^™) human breast cancer cells were grown in DMEM, high glucose, pyruvate (Gibco) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher). MCF-12A (ATCC^® CRL-10782^™) human breast epithelial cells were grown in DMEM/F12 (1:1) medium (Gibco), supplemented with 10% horse serum (Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (Thermo Fisher), 2.5 mg/ml insulin (Invitrogen), 150 μg/ml cholera enterotoxin (Sigma-Aldrich), 2.5 mg/ml hydrocortisone (Sigma-Aldrich) and 20 ng/ml epidermal growth factor (Sigma-Aldrich). Bag-1 knockout MCF-7 cells were generated using CRISPR-Cas9 system [9 (link)]. Cells were maintained at 37°C, 5% CO₂ in a humidified incubator. 60–70% confluent cells were transfected with plasmids by using IN-fect in vitro transfection reagent (iNtRON Biotechnology) according to the manufacturer’s protocol. Cells were lysed 48 hours after plasmid transfection for protein purification or immunoblotting assays.

Nonsilencing control siRNA and the siRNAs targeting mouse NLRP3 and Atg5 were chemically synthesized (Genolution Parmaceuticals Inc., Seoul, Korea) (Table 2). To knockdown endogenous NLRP3 and Atg5, cells were transiently transfected with siRNAs for 24 h at a final concentration of 100 nM and 50nM, respectively, using the iN-fect™ in vitro Transfection Reagent (iNtRON Biotechnology, Inc. Sungnam, Korea) according to the manufacturer's protocol.

On the basis
of the experimental outcomes of the miR expression profile analysis
in Vero cells in the current study, the notably highly expressed miR-1307
was selected for further targeted inhibition experiments. Anti-miR-1307
treatment alone or in combination with the anti-miR-3611 and anti-miR-8066
(mix-100 nM concentrations) treatment from IDT (Integrated DNA Technologies,
Leuven, Belgium) was performed on the Vero cells, which were infected
with SARS-CoV-2 (amount of virus used: 100 TCID50 SARS-CoV-2). Briefly,
anti-miR transiently transfected cells were seeded at 1 × 10⁴ density for 72 h and then used for further experiments detailed
below. Vero cells were seeded in a 96-well plate at a density of 1
× 10⁴ cells per well. Upon cells reaching 70–80%
confluency, the miR-1307 inhibitor transfection was performed 24 h
before virus infection. In the miR inhibitor transfection, 0.1 μL
of miR inhibitor (100 μM stock) and 0.3 μL of iN-fect in vitro transfection reagent (INtRON Biotechnology, Gyeonggi-do,
South Korea) were mixed for each well. Samples were incubated at room
temperature for 15–20 min. After incubation, the mixture was
made up to 100 μL with a nonserum medium added to each well.
Then, 100 μL of the sample was added dropwise onto the cells
from which the serum medium was removed.

The iN-fect™ in vitro Transfection Reagent (iNtRON Biotechnology, Seongnam, Korea) was used for the transfection for RNA interference to knock down c-Met expression. HepG2 cells (5 × 10⁴ cells/well) were transfected with c-Met siRNA (100 nM) or scrambled siRNA (100 nM) (SN-1002; BIONEER, Daejeon, Korea) for 48 h in serum-free media. After transfection, cells were pre-treated with GGC (30 μM) for 2 h and treated with HGF (50 ng/mL).

HCT-116 (5 × 10⁴ cells/well) cells were seeded on a 12-well plate in a medium without serum and transfected with MnSOD siRNA and scrambled-siRNA (50 nM) for 24 h by iN-fect™ in vitro Transfection Reagent (iNtRON Biotechnology, Seongnam, Korea). After transfection, cells were treated with TMP (10 μM) for 24 h.

HCT-116 (5 × 10⁴ cells/well) cells were seeded on a 12-well plate in a medium without serum and transfected with pcDNA3-MnSOD and pcDNA (300 ng) for 24 h by iN-fect™ in vitro Transfection Reagent (iNtRON Biotechnology, Seongnam, Korea). After transfection, cells were treated with TMP (10 μM) for 24 h or 6 h.