Random oligonucleotide primers

The RAW 264.7 macrophage cells were seeded in a culture dish at a density of 5 × 10⁵/mL. After incubation for 6 h, cells were pretreated with 12.5, 25, and 50 μM of TCMB (11) for 1 h and then stimulated with LPS 100 ng/mL for 6, 12, and 24 h. The cultured media were removed and cells were washed with PBS. Total mRNA was extracted by using Easy Blue^® kits (Intron Biotechnology, Seoul, Republic of Korea) and synthesized to cDNA using 0.5 mg/mL random oligonucleotide primers (Promega, Madison, WI, USA) and TOPscriptTM RTDryMIX (Enzynomics, Daejeon, Republic of Korea). PCR amplification was analyzed by using the incorporation of SYBR green (TaKaRa, Shiga, Japan) and primers for iNOS (forward strand 5′-AAC ATC CTG GAG GAA GTG GG-3′; reverse strand 5′-GCT GTG TGG TGG TCC ATG AT-3′), COX-2 (forward strand 5′-TGC TGT ACA AGC AGT GGC AA-3′; reverse strand 5′-GCA GCC ATT TCC TTC TCT CC-3′), TNF-α (forward strand 5′-AGC ACA GAA AGC ATG ATC CG-3′; reverse strand 5′-CTG ATG AGA GGG AGG CCA TT-3′), IL-1β (forward strand 5′-ACC TGC TGG TGT GTG ACG TT-3′; reverse strand 5′-TCG TTG CTT GGT TCT CCT TG-3′), and IL-6 (forward strand 5′-GGG ACT GAT GCT GGT GAC AA-3′; reverse strand 5′-CCA CGA TTT CCC AGA GAA CA-3′). All primers were purchased from Bioneer (Seoul, Republic of Korea).

The skin tissues of mice were collected, and the tissues were homogenized in Easy Blue^® kits (Intron Biotechnology, Seoul, Korea) for RNA extraction. The cDNA was synthesized using 0.5 mg/mL random oligonucleotide primers (Promega, Madison, WI, USA) and TOPscript^TM RT DryMIX (Enzynomics, Daejeon, Korea). The expression of the target genes was analyzed by measuring the incorporation of SYBR (SYBR Premix Ex Taq, TakaRa, Shiga, Japan) relative to the internal control β-actin using the Takara thermal cycler device. The PCR primers (Table S1) were designed using the Primer 3 program and custom-made in Bioneer (Daejeon, Republic of Korea)

The mRNA levels of HaCaT keratinocytes were analyzed by qRT-PCR using the LightCycler 96 Instrument (Roche Molecular Systems Inc., Basel, Switzerland) as our previous report [10 (link),49 (link),50 (link)]. Total cellular RNA in HaCaT cells was extracted by using Easy Blue^® kits (Intron Biotechnology, Seoul, Korea), synthesis to cDNA using 0.5 mg/mL random oligonucleotide primers (Promega, Madison, WI, USA) and TOPscript^TM RTDryMIX (Enzynomics, Daejeon, Korea). PCR amplification was analyzed by using the incorporation of SYBR green (TaKaRa, Shiga, Japan) and specific primers. Primer sequences are listed in Table S1.

Dulbecco’s modified Eagle’s minimum essential medium (DMEM), fetal bovine serum (FBS), fetal calf serum (FCS), penicillin, and streptomycin were obtained from Gibco (Grand Island, NY). Monoclonal antibodies against ARG-1, TMEM26, PPARγ and β-actin were from Santa Cruz Biotechnology (Delaware, CA). Antibodies against UCP-1 and PPARδ were from Bioworld Technology (Minneapolis, MN). Peroxidase-conjugated secondary antibodies were from the Jackson Laboratory (Sacramento, CA), and enzyme immunoassay (EIA) kits for catecholamines were from R&D Systems (Minneapolis, MN). Random oligonucleotide primers and M-MLV reverse transcriptase were from Promega (Fitchburg, WI). TOPscriptTM RT Dry MIX was from Enzynomics (Daejeon, Korea). Thunderbird SYBR qPCR Mix was from Toyobo (Osaka, Japan). ARG-1, MRC-1, IL-10, UCP-1, Cited1, COX7a1, TMEM26 and GAPDH oligonucleotide primers were from Bioneer (Daejeon, Korea). Phenylmethylsulfonyl fluoride (PMSF), GW9662, GSK0660, telmisartan, triiodothyronine (T3) and all other chemicals were from Sigma Chemical (St. Louis, MO).