Gapdh

Total RNA was isolated from WJ-MSCs using an AccuPrep Universal RNA Extraction kit (Bioneer), according to the manufacturer's protocol. Real-time qRT-PCR was performed with a QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 2x Power SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA) using the following program: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 56°C for 30 s, and 72°C for 30 s. Primers were purchased from Bioneer Corporation, and the primer sequences were as follows: AURKA, forward 5′-GGCCACTGAATAACACCCAAA-3′ and reverse 5′-AGAGGGCGACCAATTTCAAAG-3′; DOCK2, forward 5′-TTTCAACACCGTTCTGGAGG-3′ and reverse 5′-TCAGCGTTCTTAGGATTGGC-3′; and GAPDH, forward 5′-GAAGGTGAAGGTCGGAGT-3′ and reverse 5′-TGGCAACAATATCCACTTTACCA-3′. All PCR reactions were performed in triplicate, and comparative quantification of each target gene was normalized to GAPDH expression using the 2^-ΔΔCt method.

Methanol extraction of P. attenuatum (Pa-ME) was purchased from the Plant Extract Bank in the Plant Diversity Research Center (Daejeon, Republic of Korea; http://extract.kribb.re.kr, e-mail: mplantext@kribb.re.kr). RAW264.7 cells, a transformed macrophage cell line derived from the BALB/c mouse (ATCC number TIB-71), were purchased from ATCC (Rockville, MD, USA). Dimethyl sulfoxide (DMSO), L-N^G-nitroarginine methyl ester (L-NAME), indomethacin, lipopolysaccharide (LPS, Escherichia coli 0111:B4), pam3CSK, and (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Poly I:C was obtained from Calbiochem (La Jolla, CA). The enzyme immune assay (EIA) kits used to quantitate the levels of PGE₂ were purchased from Amersham (Little Chalfont, Buckinghamshire, UK). Specific PCR primers for iNOS, TNF-α, COX-2, and GAPDH were synthesized from Bioneer Inc. (Daejeon, Republic of Korea). Antibodies that specify phosphorylated and total forms of p65, p50, c-Jun, c-Fos, Lamin A/C, IκBα, IKKα/β, AKT, Src, Syk, ERK, p38, JNK, IRAK1, IRAK4, TAK1, MKK3/6, and β-actin were obtained from Cell Signaling (Beverly, MA, USA).

Isobutyl methyl xanthine (IBMX), dexamethasone (DEX), insulin, Oil red O, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified eagle’s medium (DMEM), bovine serum (BS), heat-inactivated fetal bovine serum (FBS), and penicillin–streptomycin (PS) were purchased from Life Technologies, Inc. (Grand Island, NY, USA). Antibodies for phospho-adenosine monophosphate-activated protein kinase α (p-AMPKα), AMPK, phospho-acetyl-CoA carboxylase (p-ACC), ACC, extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), PPARγ, C/EBPα, and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were obtained from Jackson Immuno Research laboratories, Inc. (West Grove, PA, USA). SYBR^TM Green PCR Master Mix was purchased from Applied Biosystems^TM (Foster City, CA, USA). Oligonucleotide primers of PPAR-γ, SREBP1, C/EBPα, and GAPDH were purchased from Bioneer (Daejeon, Chungbuk, Republic of Korea).

RAW264.7 cells from mice (BLAB/c, ATCC number TIB-71) were purchased from ATCC (Rockville, MD, USA). Dimethyl Sulfoxide (DMSO), L-N^G–nitroarginine methyl ester (L-NAME), lipopolysaccharide (LPS, Escherichia coli 0111:B4), and (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Gene specific PCR primers for iNOS, TNF-α, COX-2, and GAPDH were synthesized from Bioneer Inc. (Daejeon, Republic of Korea). Antibodies to phosphorylated and total protein (p65, p50, IκBα, IKKα/β, Syc, Syk, STAT3, JAK, and β-actin) were obtained from Cell Signaling (Beverly, MA, USA).

Total RNAs from cells were extracted using the RiboEx Total RNA solution (GeneAll Biotechnology, Seoul, Korea). Cells in 6-well plates (0.5 mL, 1 × 10⁵ cells per well) were transferred to tubes, to which chloroform (0.1 mL) was added, and shaken vigorously for 30 s. Aqueous phases were transferred to fresh tubes, and an equal volume of isopropanol was added. Samples were then incubated for 15 min at 4 °C and centrifuged at 12,000× g for 15 min at 4 °C. After discarding the supernatants, RNA pellets were washed once with 0.5 mL of 75% ethanol, vortexed briefly, and centrifuged at 7500× g for 5 min at 4 °C. Pellets were dried for 10–15 min and dissolved in diethyl pyrocarbonate (DEPC)-treated water. Complementary DNA (cDNA) was synthesized from the total RNA (2 µg) using SuPrime Script RT Premix with random hexamer cDNA Synthesis Kit (GeNet Bio, Daejeon, Korea), in accordance with the manufacturers’ instructions. Quantitative qRT-PCR amplification was performed using SensiFAST^TM SYBR^® No-ROX dye (Bioline, London, UK) on the CFX Connect System (Bio-Rad Laboratories, Hercules, CA, USA). The primers specific to TRP-1, TRP-2, tyrosinase, or GAPDH were purchased from Bioneer Inc. (Daejeon, Korea). Relative gene expressions were assessed using GAPDH as the internal control. The primer sequences used in this study are shown in Table 1.

Oligonucleotide primers specific for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), SREBP-1c, C/EBPα, PPARγ, UCP-1, αSMA, and Col1α1 were purchased from Bioneer (Daejeon, Korea). M-MLV reverse transcriptase and random Oligonucleotide primers were from Promega (Fitchburg, WI, USA). TOP script RT Dry MIX was from Enzynomics Co., Ltd. (Daejeon, Korea). Thunderbird SYBR quantitative polymerase chain reaction (qPCR) Mix was from TOYOBO Co., Ltd. (Osaka, Japan). All other chemicals were from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).