Penicillin

HEK293T cells, LNCaP cells, MCF7 cells, and A549 cells were purchased from Korea Cell Line Bank (KCLB, Daejeon, Korea). HEK293T cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM), and LNCaP cells, MCF7 cells, and A549 cells were cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) containing 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin (GenDEPOT, Katy, TX, USA). All the cells were maintained at 37 °C in 5% CO₂. For plasmid transfection, 2 M CaCl₂ and 2X HBS buffer (50 mM HEPES, 10 mM KCl, 12 mM glucose, 280 mM NaCl, 1.5 mM Na₂HPO₄, pH 7.05) were used in HEK293T cells, and Effectene (Qiagen, Hilden, Germany) was used in LNCaP cells following the manufacturer’s instructions. For siRNA transfection, Lipofectamine^TM 3000 (Invitrogen, Waltham, MA, USA) was used following the manufacturer’s instructions.

A549 (human epithelial cells, ATCC-CCL-185; ATCC, Manassas, VA, USA) and MRC-5 (human fetal lung fibroblast cells; ATCC-CCL-171) were cultured in TCM consisting of RPMI-1640 culture medium (GenDEPOT, Katy, TX, USA) or MEM culture medium with 100 U/mL penicillin (GenDEPOT) and 100 μg/mL streptomycin (Gibco, Carlsbad, CA, USA). The cells were maintained in a humidified incubator with 5% CO₂ at 37 °C. The A549 and MRC-5 cells were stimulated in TCM with 0.1% fetal bovine serum (FBS; Thermo Fisher Scientific, Rockford, IL, USA) and 5 ng/mL TGF-β1 (R&D Systems, Minneapolis, MN, USA) to induce EMT and FMT.

Human epidermal keratinocytes (HaCaT; CLS GmbH, Eppelheim, Germany) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GenDEPOT, Katy, TX, United States) containing 10% fetal bovine serum (FBS; GenDEPOT), 100 U penicillin, and 100 μg/ml streptomycin (GenDEPOT). The cells were then incubated in a humidified incubator with 5% carbon dioxide (CO₂)/95% air. Cells (1 × 10⁴ cells) were plated in a 96-well plate (SPL Life Sciences, Pocheon, Korea) and stabilized for 24 h. The dried EJ-AuNPs or EJ was diluted with serum-free medium (SFM) at concentrations of 25, 50, and 100 μg/ml. After the cells were washed twice with phosphate-buffered saline (PBS), diluted EJ-AuNPs solution was added to the cells and incubated for a further 24 h. To compare the cytotoxic effect of EJ-AuNPs, only SFM and commercial dexamethasone (20 μg/ml)-containing SFM were added to the cells. The cytotoxic effects of EJ-AuNPs against HaCaT cells were measured using a conventional 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, MO, United States) and a live/dead cell staining kit (Thermo Fisher Scientific, Cambridge, MA, United States), according to the manufacturer’s instructions.