Streptomycin

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.

NCI-H358 human non-small cell lung cancer cells (Korea Cell Line Bank, Seoul, Korea) were cultured routinely in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone), 2 mM glutamine (Sigma-Aldrich, St. Louis, MO, USA), 100 U/mL penicillin (Hyclone), and 100 μg/mL streptomycin (GenDEPOT, Barker, TX, USA) at 37 °C with 5% CO₂ in a humidified atmosphere.

The parental ID8 cell line was obtained from Katherine Roby (University of Kansas Medical Center, Kansas City, Kansas, USA). ID8 cells that stably express RFP or turboGFP (tGFP) have been previously described (7 (link)). The parental MC-38 cell line was obtained from Ronald DePinho (University of Texas MD Anderson Cancer Center) with permission from Jeffrey Schlom (National Cancer Institute). ID8 and MC-38 cells were cultured in DMEM supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin (GenDEPOT). To generate MC-38 cells that stably express tGFP, MC-38 cells were transfected with pGFP-V-RS vector (Origene) by using Lipofectamine 3000 reagent (Invitrogen), followed by selection with 2 μg/mL puromycin (Sigma-Aldrich).

Primary human PDLFs were prepared as described by Somerman et al. [77 (link)]. Collection of human PDL from the premolar and third molar was approved by the Institutional Review Board of Chonnam National University Dental Hospital (Approval No., CNUDH-2016-013; 20 October 2016). All participants were adults without periodontal disease. The PDLFs were grown in Dulbecco’s modified Eagle’s medium (Gibco BRL, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (GenDepot, Katy, TX, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (GenDepot, USA) at 37 °C in a humidified atmosphere containing 5% CO₂. When they achieved confluence, the cells were trypsinized using 0.25% trypsin/0.02% ethylenediaminetetraacetic acid solution (Sigma-Aldrich, St. Louis, MO, USA). Cells in which PDLFs proliferated sufficiently were sub-cultured five to six times at a ratio of 1:2 to 1:3 in a 100 mm culture dish and were used in this experiment.

Lenti-X 293T (Clontech, 632180) and MCF-7 (Korea Cell Line Bank, 30022) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GenDepot, CM-002) supplemented with 10% fetal bovine serum (FBS; Gibco, 16000-044) and 100 U/ml penicillin + 100 μg/ml streptomycin (GenDepot, CA005). The cells were grown in a humidified incubator containing 5% CO₂ at 37 °C.