Gene specific primers

Total RNA was purified from cultured cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For first-strand cDNA synthesis, 1 µg total RNA was transcribed to cDNA using a reverse-transcription system with random hexamers (A3500; Promega Corporation) according to the manufacturer's protocol. RT-qPCR was performed on a StepOnePlus Real-Time PCR system with Power SYBR-Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The PCR was performed with 1 µl cDNA in a 20 µl reaction mixture comprising 10 µl Power SYBR Green PCR Master Mix, 2 µl primers and 7 µl PCR-grade water. The PCR program was as follows: A denaturation step at 95°C for 10 min, 40 cycles each of 95°C for 15 sec and 60°C for 1 min. Quantification of gene expression data was calculated using the 2^−ΔΔCq method the crossing point of the target genes with β-actin was calculated using the formula 2-^{(target gene−β−actin)} and the relative amounts were quantified (39 (link)). The sequences of the gene-specific primers used (Bioneer Corporation, Daejeon, Korea) are listed in Table I.

Extraction solvents were acquired from Samchun [Seoul, Korea: methylene chloride, D1602; methanol (MeOH), M1450], Junsei Chemical Co., Ltd. [Tokyo, Japan: n-butanol (n-BuOH), 6313050380], and JT Baker (Philipsburg, NJ, USA: n-hexane, 9304-03). Cell culture and assay reagents, Oil red O stain, and staining solvents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Gene specific primers were from Bioneer (Daejeon, Korea) and the primary antibodies were purchased from Cell Signaling Technology, Inc. [Danvers, MA, USA: PPARγ, #2443; CCAAT/enhancer-binding protein (C/EBP) α, #2295; p-38, #4511; p-c-Jun N-terminal kinase (JNK), #9255; p-extracellular-signal-regulated kinase (ERK), #4370], Abcam [Cambridge, UK: sterol regulatory element-binding protein-1c (SREBP-1c), ab3259], and Santa Cruz Biotechnology (Dallas, TX, USA: β-actin, sc-47778). Sources for other reagents and equipment were given in the designated sections when they are first mentioned.