Quantitative RT-PCR Analysis of Epithelial-Mesenchymal Transition Markers

These procedures were performed as previously described^{5 (link),17 (link)}. Briefly, total RNA was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. First-strand cDNA was synthesized via reverse transcription using the Revert Aid^TM First Strand cDNA Synthesis Kit (cat. no. 6210A; TaKaRa Bio, Inc., Otsu, Japan). Subsequently, qRT-PCR was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and SYBR^® Green mix (Tiangen Biotech Co., Ltd., Beijing, China). The qRT-PCR thermal cycling conditions were initiated using a denaturation step at 95 °C for 15 min and comprised 40 cycles (denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and elongation at 72 °C for 30 s). The amplification products were analyzed using the 2^−∆∆Cq method^{18 (link)}, and the expression levels in each sample were calibrated to those of the housekeeping gene GAPDH. The primers employed for amplifying HDAC4, E-cadherin, N-cadherin, Snail, Slug, and GAPDH are listed in Supplementary Table 1.