First stand reverse transcription system

The information of antibodies was as followed: ACAT1 (1:1000, HPA004428, Sigma, USA), β-catenin (1:1000, sc-376841, Santa Cruz, USA) and E-cadherin (1:1000, #3195P), Vimentin (1:1000, #5741P) and GAPDH (1:10000 #5174P) were purchased from Cell Signaling Technology.
The demethylating reagent 5-aza-dC was purchased from Sigma (#A3656, USA). Full-length cDNA from the open reading frame of ACAT1 (Origene, USA) was subcloned into the pCMV6-Entry vector (Origene, USA). NPC cells were cultured in 6-well dishes to 70-90% con uence then transfected with 2μg pCMV6-Entry or ACAT1 plasmid using an X-treme GENE HP DNA Transfection Reagent (Roche, Germany) for 48 hrs.
Real-time RT-PCR Brie y, rst-strand complementary DNA was synthesized using a First-Stand Reverse Transcription System (Transgene, Beijing, China). Real-time RT-PCR was carried out using SYBR Green PCR master mix in Step One Plus System (Applied Biosystems, USA). The primer sequences and cycling conditions for all experiments were as follows, ACAT1-F 5'-GGCTGGTGCAGGAAATAAGA-3', ACAT1-R 5' GGAATCCCTGCCTTTTCAAT-3'; GAPDH-F 5'-GCTCAGACACCATG-GGGAAG-3', GAPDH-R 5'-TGTAGTTGAGGTCAATGAAGGGG-3'. Empty vector-transfected cells were used as control. The relative gene expression was calculated using the comparative threshold cycle (2 -△△CT ) equation. All the experiments were performed in triplicate.