Sybr probe

For qRT-PCR analysis, total RNA was extracted from 7-day-old seedlings using the RNeasy plant mini kit (Qiagen), and the first-strand cDNA was synthesized using the Takara PrimeScript First-strand cDNA synthesis kit (TAKARA). Next, PCR was performed on an Illumine Eco (Illumina) System with an SYBR probe (TAKARA). Expression levels of eIF4E1 were normalized to the expression level of two reference genes (ACTIN 2 and TUBULIN 2), respectively. The primers used are listed in Supplementary Table S1. Results obtained from three biological repeats with standard deviation (SD).

Total RNA were extracted from the serum-starved mNSCs and embryonic brain tissues (E 13.5, n = 3) using the RNeasy R Micro Kit (Qiagen, Germany). The quantity of RNA was analyzed with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and then reversely transcribed into cDNA system. qRT-PCR was performed using an SYBR probe (Takara Bio, Kusatsu, Japan), and fluorescence was detected with a CFX96 Connect Real-Time PCR Detection System (Biorad, Hercules, CA, USA). BMP2/4, Smad1/5/8 and Runx2 (runt-related transcription factor 2) were the key genes of the BMP/Smad signaling pathway. The primers were purchased from Sangon Biotech (Shanghai, China), and the sequences are listed in Table 1. The reaction was carried out in a 25 μL system. After the reaction, the relative expression of the target gene was calculated according to the average value of threshold cycle (Ct value) of the target gene and the housekeeping gene, which were expressed as 2^−△△Ct.