Gene Expression Quantification Protocol

These procedures were performed as previously described^{20 (link),23 (link)}. Briefly, total RNA was isolated using TRIzol^® Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. Reverse transcription for first-strand cDNA was synthesized using the RevertAid^TM First Strand cDNA Synthesis Kit (cat. no. 6210A, TaKaRa Bio, Inc., Otsu, Japan). Subsequently, qPCR was performed in a CFX96 Real-Time PCR Detection system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using SYBR^® Green mix (Tiangen Biotech Co., Ltd., Beijing, China). The qPCR thermal cycling conditions were as follows: a denaturation step at 95 °C for 15 min and 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 30 s and elongation at 72 °C for 30 s. The amplified products were examined using the 2^−∆∆Cq method^{24 (link)}, and each sample was calibrated to the expression levels of the housekeeping gene GAPDH. The primers are shown in Supplementary Table 1.