48 well plate

To assess the activity of isoegomaketone against intracellular M. abscessus, BMDMs and differentiated THP-1 cells (1.5 × 105 cells/well) were dispensed into 48-well plates (SPL, New York, NY, USA) and then infected with the reference or clinically isolated M. abscessus strains at a multiplicity of infection (MOI) of 1:1. After 4 h of infection, the cells were washed with PBS to remove the uninfected bacteria and incubated with DMEM containing 20 μg/mL amikacin for 1.5 h to kill the remaining extracellular M. abscessus. The cells were then washed with PBS and a broth medium with various concentrations of isoegomaketone was added. After 24 h or 48 h of incubation at 37°C and 5% CO2, BMDMs and differentiated THP-1 cells were extensively washed with PBS, lysed with 0.05% Triton X-100, and then serially diluted with PBS. The number of CFUs was quantified by plating serial dilutions of lysates on M7H10 agar plates.

Bacteria grown for 16 h in culture were used to take inoculum and diluted by 1:1000 in fresh YCFAm broth and cultivated. When necessary, the medium was supplemented with ½ MIC of E. coli of each metal(oid) toxicant tested in 48-well plates (SPL life sciences) at 37 °C for 18 h with orbital shaking. The dynamics of grown bacteria were estimated by optical density at 600_nm.

RAW264.7 cells were cultured at 1 × 10⁵/well in 48-well plates (SPL life sciences Co., Pocheon, Korea) for 24 h. The cells were washed with fresh medium and treated with ChondroT or GHJTY (1, 0.3, or 0.1 mg/mL) for 2 h, followed by treatment with 500 ng/mL LPS (Sigma Co., MO, USA) for 24 h. IL-6 (Biolegend, San Diego, CA, USA), TNF-α (R&D system, Minneapolis, MN, USA), IL-1β (R&D System, Minneapolis, MN, USA), and PGE₂ (R&D system, Minneapolis, MN, USA) in the supernatants were measured using ELISA kits following the manufacturer’s experimental protocols. The assay was performed at room temperature and the optical absorbance was measured at 450 nm using an ELISA microplate reader (ELx808) within 30 min.

RAW 264.7 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and was grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich; St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé France), and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin; Gibco; Waltham, MA, USA) at 37°C in the presence of 5% CO₂. RAW 264.7 cell (3 x 10⁴ cells per well) was seeded on 48-well plates (SPL Life Sciences, Pocheon, Republic of Korea) and incubated for 16 h. Attenuated Salmonella candidates were cultured in LB at OD₆₀₀ of 1.0 and harvested. The strains were treated to RAW 264.7 cells at multiplicity of infection (MOI) of 10 and incubated for 2 h at 37°CC. The cells were washed with PBS (Welgene, Gyeongsan, Republic of Korea) thrice and transferred to DMEM supplemented with 10% FBS and 100 μg/mL of gentamicin to eliminate extracellular bacteria. After 2 h incubation, the cells were washed three times with PBS and treated with RIPA lysis buffer (Sigma) for invasion assay. For replication assay, the cells were incubated with DMEM supplemented with 10% FBS and 10 μg/mL gentamicin to prevent the leakage of intracellular bacteria. After 16 h incubation, cells were treated with RIPA lysis buffer (Sigma). Subsequently, lysis samples were serially diluted with PBS and spotted on the LB agar plate.

MCF7 cells were seeded in 48-well plates (SPL Life Sciences, Pocheon-si, South Korea). After complete adhesion, cells were transfected with 1 µg of vector (pEGFP-1-p1 or pEGFP-1-p2) using Lipofectamine LTX & PLUS reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. pEGFP-1 is a promoterless vector containing an eGFP reporter. The presence of the GFP signal was monitored in MCF7 cells transfected with pEGFP-C1 as the positive control and pEGFP-1-p1 or pEGFP-1-p2 vectors 48 h after transfection and under a fluorescent microscope.

RAW264.7 cells were seeded into 48-well plates (SPL Life Sciences, Pocheon, South Korea) at 2 × 10⁵ cells/mL and treated with ZCO (0.0025, 0.005, and 0.01%) or the positive control Bay11-7082 (20 μM) for 2 h before stimulation with 500 ng/mL of LPS. The supernatants were collected after incubation for 24 h to measure the TNF-α (R&D system, Minneapolis, MN, United States) and IL-6 (Biolegend, CA, United States) with an ELISA kit according to manufacturer’s instructions.