Sybr premix

The Bio-Rad Real-time PCR system CFX96 and SYBR Premix (TranStart Green qPCR SuperMix, TransGen Biotech) were used to quantify gene expression. Total RNA extraction (EasyPure Plant RNA Kit, TransGen Biotech) and qRT-PCR were carried out according to the RNA extraction and SYBR Premix instructions, respectively. The specific primer pairs used are listed in Supplementary Table 2. Data were analyzed by the Livak method (Livak and Schmittgen, 2001 (link)) and expressed as a normalized relative expression level (2^-ΔΔCT) of the respective genes, and the relative transcript level of each sample was normalized to actin (Supplementary Table 2). Six biological replicates were included in the experiment. The IBM SPSS version 21 software was used for statistical analysis, the p value was determined using Fisher’s protected LSD test.

Total RNA was extracted from cell samples using TRIzol Reagent (Invitrogen) and quantified using the NanoDrop 2000 spectrophotometer (Thermo Scientific, Bremen, Germany). The first strand of cDNA was synthesized using M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s instruction. Oligo (dT) was used as the primers for reverse transcription of mRNA. Stem-loop RT primers were used for the reverse transcription of miRNAs. Actin and U6 were used as their respective controls. Semi-quantitative PCR and real-time quantitative PCR were performed in ABI PCR machines using pre-stained mix (Tiangen) and SYBR Premix (TransGen Biotech) respectively. The primers used for reverse transcription and RT-PCR were listed in Table 1.Table 1

Primers used for reverse transcription and RT-PCR.

Name	Sequences
FABP4-F	ACTGGGCCAGGAATTTGACG
FABP4-R	CTCGTGGAAGTGACGCCTT
PPARγ-F	GCTGACCAAAGCAAAGGCG
PPARγ-R	GCCCTGAAAGATGCGGATG
LPL-F	TCATTCCCGGAGTAGCAGAGT
LPL-R	GGCCACAAGTTTTGGCACC
PLIN1-F	GCGAGGATGGCAGTCAACAAA
PLIN1-R	GCACGCCCTTCTCATAGGCAT
Actin-F	CATGTACGTTGCTATCCAGGC
Actin-R	CTCCTTAATGTCACGCACGAT
miR-301b-RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTTTGA
miR-130b-RT	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACATGCCCT
miR-301b-Up	GCGGCGCAGTGCAATGATATT
miR-130b-Up	GTCGTGCAGTGCAATGATGAAA
miRNA-Dn	TCCAGTGCAGGGTCCGAGGT
U6-RT	AAAATATGGAACGCTTCACGAA
U6-Up	CTCGCTTCGGCAGCACATATA
U6-Dn	ACGCTTCACGAATTTGCGTGTC