Lm001 05

All cell culture reagents were purchased from Welgene (Gyungsan, Korea). Hep3B human hepatocyte cancer cell and Raw 264.7 mouse kupffer cell were maintained at 37 °C in a 5% CO2 atmosphere in DMEM (Welgene, LM001–05) supplemented with 5% (vol/vol) fetal bovine serum, penicillin (100 U/mol) and streptomycin (100 μg/ml). For experiments, cells were maintained in a steroid-free condition with phenol free DMEM (Welgene, LM002–05) media containing 2% charcoal dextran treated-FBS, penicillin (100 U/mol) and streptomycin (100 μg/ml). Genistein (LC laboratories, G-6055) and cobalt(II) chloride hexahydrate (Sigma, C8661) were added every 12 h with media change. Lipopolysaccharides (LPS, Sigma, L6511) was added after dissolved in distilled water (100 ng/ml). All cell experiments were repeated at least 3 times.

After H9c2 cells were cultured with DMEM-High glucose (LM 001-05, Welgene) added to 5% FBS and 1% P/S, the medium was changed to a low glucose medium (LM 001-17, Welgene), which contained glucose 1000 mg/L, and was added to 2% CD-FBS and 1% P/S, similar to the above starvation medium. Following additional supplementation with fatty acids (Palmitic acid 330 μM, Oleic acid 660 μM), the cells were incubated for 48 h. At 24 h of incubation with the low glucose medium, extra glucose was supplemented to high glucose group cells using 45% D-glucose (LS 001-02, Welgene) to reach glucose concentrations of 4500 mg/L, and incubation was maintained for the remaining 24 h.

The C2C12 cells (CRL-1772, ATCC^®, Manassas, VA, USA) were incubated at 37 °C in 5% CO₂. The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; LM001-05, Welgene, Gyeongsangbuk-do, Republic of Korea) containing 10% fetal bovine serum (S001-07, Welgene) and 1% ZellShield™ (#13-0150, Minerva-Biolabs, Berlin, Germany). To differentiate C2C12 myoblasts into C2C12 myotubes, the cells were seeded at 2.5 × 105 cells/well in a 6-well plate and cultured at full density for 2 days. The medium was then replaced with a differentiation medium containing 2% horse serum (#16050-122, Thermo Fisher Scientific, Waltham, MA, USA) and 1% ZellShield™ and changed daily for 5 days. To determine the effect of the NNL extract on the DEX-induced cell damage, the differentiated C2C12 myotubes were treated with 100 μM DEX and 50, 100, and 200 μg/mL NNL extract for 24 h.

ES-E14TG2a, a mouse embryonic stem cell line (mESCs), was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured using the same method described elsewhere [29 (link)]. The composition of the growth medium for maintaining undifferentiated cells was as follows: DMEM/F-12 (1:1) (11320-033, Gibco, Grand Island, NY, USA), 10% FBS (16000-044, Gibco, Grand Island, NY, USA), non-essential amino acid (NEAA; 11140-050, Gibco, Grand Island, NY, USA), plasmocin prophylactic (Plas; ant-mpp, InvivoGen, San Diego, CA, USA; 50 μg/mL), 100 U/mL penicillin and 100 mg/mL streptomycin (P/S; L0022, Biowest, Riverside, MO, USA), 2-mercaptoethanol (21985-023, Gibco, Grand Island, NY, USA; 10⁻⁴ M), and mouse leukemia inhibitory factor (mLIF; ESG1107, Millipore, Darmstadt, Germany; 10 ng/mL). Mouse embryo fibroblasts (mEFs) were used as the feeder cells. The mEFs were obtained from E10.5 mouse embryos [30 (link)]. The cells were grown at 37 °C in a 5% CO₂ humidified tissue culture incubator (MCO-18AIC, Sanyo, Osaka, Japan). The 3T3-L1 cells (Clone A31 and ATCC) for the general toxicity test were cultured in DMEM (LM 001-05, WELGENE, Gyeongsan-si, Gyeongsangbuk-do, South Korea) with 10% fetal bovine serum (FBS; S1480-500, Biowest, Nuaillé, France) and P/S.

HEK293T cells (Riken) were cultured in Dulbecco's modified Eagle's medium (DMEM; Caisson) containing 5% fetal bovine serum (FBS; Welgene), l‐glutamine (20 mM), and penicillin‐streptomycin (1%, Thermo Fisher). The isolation of human ORS and DP cells was performed as previous study (Messenger, 1984 (link); Limat & Noser, 1986 (link)). In brief, for ORS cell culture, the bulge region of the dissected human HFs was cutoff to prevent contamination with other cells. The trimmed bulge region was immersed in DMEM (#LM 001‐05, Welgene) supplemented with 20% FBS. On the third day of culture, the medium was changed to Epilife medium (#MEPI500CA, Gibco) supplemented with human keratinocyte growth supplement (#S0015, Gibco). For DP cell culture, the proximal parts of the HF (bulb area) were removed using a needle, and DP was isolated from the tip of hair bulbs. The DPs were immersed in DMEM supplemented with 20% FBS. The explanted DPs were left for 14 days. Then, the medium (DMEM supplemented with 10% FBS) was changed every two days. All cells were cultured at 37°C in a 5% CO₂ atmosphere.