Ccd 986sk

The cytotoxicity of the extracts was determined using the 3-(4,5-dimethythiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as follows [36 (link)]: 1.0 × 10⁴ human fibroblast cells/well (CCD-986sk, ATCC, Manassas, VA, USA) were inoculated into a 96-well plate and further incubated in a CO₂ incubator at 37°C for 6 hours. Subsequently, 200 μL of the three extracts (GE, WE, and FE) with various concentrations was added to the cells, and the cells were cultured for 24 hours. MTT solution (5 μg/mL) was added to each well, and the supernatant was removed 4 hours later. Thereafter, 10 μL of acid-isopropanol (0.04 N HCl in isopropanol) was added to the solution, and the absorbance of the solution was measured using a microplate reader at a wavelength of 565 nm. The cell cytotoxicity was expressed as the percent ratio of the cell number in the sample treated with a certain concentration to the cell number in the untreated control sample [37 (link)].
The morphology and density of the fibroblast cells treated with the various extracts at doses of 0.01% and 0.5% were also observed under an inverted microscope (IMT-2, Olympus, Tokyo, Japan) and compared with those of the control (untreated cells).

HaCaT (human keratinocytes) (Cell Line Service, Eppelheim, Germany) and human dermal fibroblasts (CCD986sk, ATCC, Manassas, VA, USA) were incubated in DMEM (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37 °C and humidified 5% CO₂ atmosphere.
Ginsenoside C-K, F1, F2, G17, G75, PPD, PPT, Rg2, Rg3, Rh1, and Rh2 (>95% pure) were prepared using enzymatic methods as previously reported [17 (link),41 (link),42 (link),43 (link),44 (link),45 (link),46 (link),47 (link)]. Ginsenosides Rb1, Rd, Re, and Rg1 were directly purified from protopanaxadiol (Hongjiu Biotech Co., Ltd., Dalian, China) type or protopanaxatriol (Da Nature Biological Engineering Co., Ltd., Fusong, China) type ginsenoside mixtures using a silica column (168 × 71 mm id, Biotage, Uppsala, Sweden), an ODS column (157 × 39 mm id, Biotage, Uppsala, Sweden), and recycling preparative HPLC (>95% purity). Ginsenosides were dissolved in dimethyl sulfoxide (DMSO). Madecassoside (MA) was purchased from Changzhou United Chemical Co., Ltd. (Changzhou, China). RU486 (GR antagonist) and DEX (GR agonist) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Control siRNA and GR siRNA were purchased from Cell Signaling Technology (Danvers, MA, USA).

Primary human epidermal keratinocytes (HEKn; American Type Culture Collection [ATCC], Manassas, VA, USA) were cultured in growth medium (GM) consisting of dermal cell basal medium (ATCC) supplemented with a keratinocyte growth kit (ATCC). Cultures were maintained at 37 °C and 5% CO₂.
Human dermal fibroblasts (CCD-986Sk; ATCC) were cultured in medium composed of Iscove’s Modified Dulbecco′s Medium (Welgene, Gyeongsan, Korea), fetal bovine serum (Gibco^TM, Thermo Fisher Scientific, Rockford, IL, USA), and 1% penicillin/streptomycin (Welgene). Cells were also maintained at 37 °C and 5% CO₂.