Trypsin

Tyrosinase from mushroom (T3824-25KU, tyrosinase activity 7164 unit/mg solid), L-3,4-dihydroxyphenlyalanine (L-DOPA, D9628), 3-(4-Hydroxyphenyl)-L-alanine (L-tyrosine, T3754), 5-hydroxy-2-(hydroxymethyl)-4H-pyran (kojic-acid), Arbutin and α-Melanocyte Stimulating Hormone (α-MSH, M4135) were purchased from Sigma Aldrich (St, Louis, US). Dulbecco modified Eagle medium (DMEM), Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS), and Trypsin were purchased from WELGENE (Gyeongsangbuk-do, Korea). Cell Viability Assay Kit (EZ-Cytox) was purchased from DOGEN (Seoul, Korea).

Primary astrocytes were isolated from the cerebral cortices of Sprague Dawley (SD) rats on postnatal days (PND) 1–2 (Daehan Biolink Co. Ltd., Chungbuk, Korea). Briefly, cortices were dissected and chopped in ice-cold Hanks’ balanced salt solution (HBSS) buffer (Welgene Inc., Daegu, Korea). To isolate cells from debris, tissues were then treated with 0.25% trypsin (Welgene Inc., Daegu, Korea) for 30 min at 37 °C, washed with HBSS, mechanically dissociated, and plated in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12; Gibco, Waltham, MA, USA) medium containing 10% fetal bovine serum (FBS; Welgene, Daegu, Korea) on poly-L-lysine coated plates. Cultures were maintained under the same conditions and used for experiments after 14–18 days in vitro.

RPMI 1640 medium, fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (DPBS), penicillin-streptomycin solution, and trypsin-ethylenediaminetetraacetic acid (EDTA) (WELGENE, Daegu, Korea); thymidylate synthase (TS, Cell Signaling Technology, Danvers, MA, USA); cyclin-dependent kinase (CDK1), cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), cyclin A, cyclin B1, cyclin D1, cyclin E (Santa Cruz Biotechnology, Dallas, TX, USA); β-actin (Thermo-Fisher Scientific, Waltham, MA, USA); a water-soluble tetrazolium salt (WST)-8-based cell viability assay kit (DoGen, Seoul, Korea); bovine serum albumin (GenDEPOT, Katy, TX, USA); secondary horseradish peroxidase (HRP)-conjugated antibodies (GeneTex, Inc., Irvine, CA, USA); n-butanol (J.T. Baker, Mexico City, Mexico); berberine, palmatine, coptisine, and jatrorrhizine (Avention, Incheon, Korea).

Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin antibiotic solution, and trypsin were obtained from Welgene (Gyeongsan, Korea). Phosphate-buffered saline (PBS) was purchased from Lonza (Basel, Switzerland). Piperlongumine was obtained from INDOFINE Chemical Company, Inc. (Hillsborough Township, NJ, USA). N-acetylcysteine (NAC), trypan blue solution, and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Amresco (Solon, OH). Dimethyl sulfoxide (DMSO) and 2′, 7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma (St. Louis, MO, USA). An NF-κB inhibitor (Bay 11-7082), and antibodies against cyclin D1, CDK4, CDK6, proliferating cell nuclear antigen (PCNA), NF-κB p65, IκBα, lamin B, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), goat anti-rabbit IgG-HRP, and donkey anti-goat IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against cyclin B1, p-CDK1, and p-IκBα were obtained from Cell Signaling Technology (Danvers, MA, USA). IκB kinase-β antibody (IKK-β) was purchased from Abcam (Cambridge, MA, USA).

HSG cells isolated from human submandibular ducts and A253 cells derived from human squamous carcinoma in submandibular glands were cultured in Dulbecco's Modified Eagle's Medium (Welgene, Daegu, South Korea) supplemented with 10% fetal bovine serum (Welgene) and 1% penicillin/streptomycin (Gibco, Carlsbad, CA, USA) at 37°C in a humidified 5% CO₂ incubator. Media change was done three times a week and sub-cultured when cells were approximately 80% confluent. 0.25% trypsin/1.0 mM ethylenediaminetetraacetic acid (Welgene) was used to detach cells from the culture dish. Primary cultured normal human fibroblast cells were obtained from biopsy sample of a patient. Normal tissue was submerged in 70% EtOH for 2 min for the sterilization and washed twice with PBS. Normal tissue was minced in HBSS and transferred to cell culture dish containing DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, and incubated for 7 days. At the day 7 media and unattached cells were completely removed. Attached cells were split into four 100mm dishes and continuously subcultured or cryopreserved. Media was changed twice a week.

Dulbecco’s Modified Eagle Medium (DMEM), trypsin, and fetal bovine serum (FBS) were purchased from Welgene (Gyeongsan, Republic of Korea). Streptomycin–penicillin was purchased from Gibco (NY, USA). Calcium-free DMEM, superscript™ IV first-strand synthesis system, and Dream taq Green PCR Mix were purchased from Thermo Fisher Scientific (MA, USA). TRIzol reagent was purchased from Invitrogen (CA, USA). Sodium dodecyl sulfate (SDS), dithiothreitol (DTT), agarose, lipopolysaccharide (LPS), ethyl ether, 1,1-diphenyl-2-picrylhydrazyl (DPPH), Griess reagent, NG-Methyl-l-arginine acetate salt (L-NMMA), and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma Aldrich (St. Louis, USA). Reagent set B, cytokine IL-6 and IL-8 ELISA sets used for immunoassay were purchased from BD Biosciences (NJ, USA). TI was acquired in a previous study [31 (link)].