Quantitative Real-Time PCR of EMT Markers

Total RNAs were extracted by RNAios Plus (9109, Takara) after which reverse transcription was carried out to synthesize cDNA as previously reported [37 (link)]. Quantitative real-time PCR (qRT-PCR) assays were carried out using a LightCycler system (Roche) with a SYBR qPCR Master Mix (Q711, Vazyme). The primers used for qRT-PCR were: 5′- CGGGAGTCCGCAGTCTTA-3′ (sense) and 5′- GCTTGAGGGTCTGAATCTTG -3′ (antisense) for Twist1, 5′- GGCTCCTTCGTCCTTCTCCTCTAC-3’ (sense) and 5′- CCTGGCACTGGTACTTCTTGACATC-3′ (antisense) for Snail (snail family transcriptional repressor 1), 5′-CCTCCTCCATCTGACACCTCCTC-3′ (sense) and 5′- AGGTAATGTGTGGGTCCGAATATGC-3′ (antisense) for Slug (snail family transcriptional repressor 2), 5′- TCTTCACCGTCTCTTTCAGCATCAC-3′ (sense) and 5′-GAGGAGAACTGGTTGCCTGTAATGG-3′ (antisense) for Zeb1 (zinc finger E-box binding homeobox 1), 5′-GGTTGAATCTGACTGACGGCTCTG-3′ (sense) and 5′-TGCCCGACTCCTGGATCACTTC-3′ (antisense) for USP13, 5′- GGGTTGAACCATGAGAAGT-3′ (sense) and 5′- GACTGTGGTCATGAGTCCT-3′ (antisense) for GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Relative mRNA levels were calculated using the 2^−ΔΔCT method [40 (link)] with GAPDH as an internal control.