Tyrosinase

α-MSH, EGC, H89, SB203580, and TRIzol reagent used in the present study were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s Medium (DMEM), fetal bovine serum, penicillin/streptomycin, L-glutamine, and trypsin-EDTA (ethylenediaminetetraacetic acid) were purchased from Hyclone Laboratories (UT, USA). Antibodies including CREB, ERK (extracellular signal-regulated kinases), JNK1 (c-Jun N-terminal kinases 1), JNK2 (c-Jun N-terminal kinases 2), laminin B, MC1R, MITF, p-CREB, TRP-1, TRP-2, and tyrosinase were purchased from Santa Cruz Biotechnology (Dallas city, TX, USA). p38 MAPK, phospho-p38 (p-p38) MAPK, phospho-JNK, PKA, and p-PKA were purchased from Cell Signaling Technology (Beverly, MA, USA). Phospho-ERK and β-actin were purchased from Novus Biologicals (Littleton, CO, USA). The second antibody conjugated with horseradish peroxidase was purchased from Santa Cruz and Sigma-Aldrich.

Primary antibodies for mouse anti-MITF (#sc-25386), tyrosinase (#sc-15341), TRP-1 (#sc-514900), TRP-2 (#sc-74439), p-ERK (#sc-7383), ERK 1/2 (#sc-514302), and β-actin (#sc-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The primary anti-bodies for mouse anti-cyclic AMP (cAMP) response element-binding protein (CREB) (8763) (#9198S) and CREB (D76D11) (#4820S) were purchased from Cell Signaling Technology (Beverly, MA, USA); all primary antibodies were used at 1:1000 dilutions. Secondary mouse antibody for primary antibodies was purchased from Enzo Life Sciences (Farmingdale, NY, USA) and used at 1:5000 dilutions. Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), and penicillin/streptomycin were purchased from Gibco BRL (Carlsbad, CA, USA). Most chemicals, including α-MSH, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), L-DOPA, 1,1-diphenyl-2-picryl-hydrazy (DPPH), 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), arbutin, and mushroom tyrosinase were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The cells were washed with PBS and centrifuged at 1000× g for 5 min at 4 °C. The obtained pellets were resuspended in RIPA buffer with protease and proteasome inhibitors, incubated on ice for 20 min, sonicated, and centrifuged at 20,000× g for 20 min at 4 °C. The supernatants were separated using 8% or 10% SDS-PAGE and then transferred to nitrocellulose membranes. The membranes were incubated overnight at 4 °C with primary antibodies against MITF, GAPDH (Thermo Fisher Scientific, Rockford, IL, USA), tyrosinase, TRP1, TRP2, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cAMP-responsive element binding protein (CREB), or pCREB (Cell Signaling Technology, Danvers, MA, USA), which was followed by incubation with the appropriate secondary antibodies (Thermo Fisher Scientific, Rockford, IL, USA). The blots were developed by using the EZ-Western Lumi Femto™ western blot detection kit (Daeil Lab Service, Seoul, Korea).

Loliolide (purity: 98% by HPLC) was purchased from Chemfaces (Wuhan, China). HaCaT and B16F10 cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), and penicillin-streptomycin were purchased from HyClone (Logan, UT, USA). 3-(4-5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco (Brisbane, Australia). 2,2’-Azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS), ascorbic acid, α-melanocyte stimulating hormone (α-MSH), TRIzol, and arbutin were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). The cDNA synthesis kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Forward and reverse primers for polymerase chain reaction (PCR) and real-time PCR were synthesized by Macrogen (Seoul, Korea), and PCR premix was purchased from Bio-D Inc. (Seoul, Korea). Polyvinylidene difluoride (PVDF) membrane was purchased from Merck Millipore (Billerica, MA, USA). Antibodies against PI3K, p-PI3K, AKT, p-AKT, Keap1, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against NRF2, HO-1, CREB, p-CREB, MITF, and tyrosinase were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

SLUG (sc-166476), Tyrosinase (sc-20035), SOX9 (sc-20095), Fra-1 (clone D-3, sc-376148), nucleolin C23 (sc-8031) and ZEB1 (sc-25388) (Santa Cruz Biotechnology, INC, Dallas, TX, USA), Snail (#3895) and p27Kip1 (#2552) (Cell Signaling Technology, Leiden, Netherlands), Microphthalmia (Ab-1, #OP126L, Calbiochem Thermo Fisher, Waltham, MA, USA), Twist (clone 2C1a #ab50887 AbCam, Cambridge, UK), PRAME (TA309818, OriGene, Rockville, MD, USA), ZEB2 (HPA003456, Atlas Antibodies) and SPARC (OSN4.2, #M124 from Takara, Kusatsu, Japan) were used in accordance to the manufacturer’s instructions. A mouse monoclonal and a rabbit monoclonal Abs were utilized against E-Cadherin (clone 36 BD #610181, Transduction Laboratories and clone 24E10, Cell Signaling Technology, Leiden, Netherlands #3195). A specific polyclonal rabbit antibody was generated against a human SCD5 synthetic peptide (aa 313-327) (Eurogentec Group, Liege, Belgium). β-actin (Clone AC-15 #A5441) and α-Tubulin (clone B-5-1-2 #T5168 (Sigma Aldrich St. Louis, MO, USA) were used as loading controls.