DPCs were seeded into a 6-well plate with 2.5 × 105 cells per well. After treating the cells with 0, 25 and 50 µmol/L Hordenine for 24 h, the total RNA was extracted with Magzol Reagent (Magen, KD210300, Guangzhou, China). Reverse-transcription PCR was conducted with 1000 ng of total RNA using a Hifair® Ⅱ 1st Strand cDNA Synthesis SuperMix (Yeasen, 11120ES60, Shanghai, China). Q-PCR was performed with Hieff® qPCR SYBR Green Master Mix (Yeasen, 11202ES03, Shanghai, China) to detect the expression of related genes according to the following conditions: 95 °C for 15 s, 55 °C for 30 s, and 72 °C for 30 s (40 cycles). β-actin was used as an internal control.
All the primers used in the real-time quantitative PCR were as follows: ALP, 5′-CCAACTCTTTTGTGCCAGAGA-3′ (forward) and 5′-GGCTACATTGGTGTTGAGCTTTT-3′ (reverse); Versican, 5′-TTTTACCCGAGTTACCAGACTCA-3′ (forward) and 5′-GGAGTAGTTGTTACATCCGTTGC-3′ (reverse); Wnt3a, 5′-CTCCTCTCGGATACCTCTTAGTG-3′ (forward) and 5′-GCATGATCTCCACGTAGTTCCTG-3′ (reverse); β-catenin, 5′-ATGGAGCCGGACAGAAAAGC-3′ (forward) and 5′CTTGCCACTCAGGGAAGGA-3′ (reverse); Lef-1, 5′-AGAAATGAGAGCGAATGTCGTAG-3′ (forward) and 5′-CTTTGCACGTTGGGAAGGA-3′ (reverse); Axin2, 5′-TGACTCTCCTTCCAGATCCCA-3′ (forward) and 5′-TGCCCACACTAGGCTGACA-3′ (reverse); Cyclin d1, 5′-GCGTACCCTGACACCAATCTC-3′ (forward) and 5′-CTCCTCTTCGCACTTCTGCTC-3′ (reverse).
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