Quantitative Real-Time PCR Protocol

Total RNA was purified from cells in 60-mm dishes using TRIzol (Invitrogen) according to the manufacturer’s instructions [35 (link)]. Then, cDNA was obtained from total RNA extracted from cells using a reverse transcription (RT) reaction kit (TAKARA, Japan) [13 (link), 36 (link)]. cDNA was completed with SYBR Premix Ex Taq™II (TaKaRa, Japan), and the program was carried out as follows: 95 °C for 30 s for one cycle; then, 95 °C for 5 s and 60 °C for 30 s, followed by plate reading for 40 cycles. The PCR primers (Table S1) were designed using Primer3 plus in Supplementary Table 1. The relative expression levels of the mRNAs in the groups were analyzed using the 2ΔΔCT method [32 (link), 37 (link)].