RNA extraction and quantitative RT-PCR analysis

The chondrocytes isolated from 6-day-old mice were seeded onto a 24-well plate and stimulated by treatment with α-mannosidase (1.9 U/mL) for 74 hr. Total RNA was extracted from the samples using an RNeasy Mini Kit (QIAGEN, Hilden, Germany). For complementary DNA synthesis, 1.0  μg of RNA was reverse transcribed using random hexamer primers (Promega, Tokyo, Japan) and ImProm II reverse transcriptase (Promega). Quantitative RT-PCR was performed using a Thermal Cycler Dice Real Time System II (Takara Bio Inc, Otsu, Japan) and SYBR Premix Ex Taq II (Takara, Shiga, Japan) with gene-specific primers (Supplementary file 1) in 15 μl of the mixture, following the manufacturer’s instructions. Quantitative data were normalized using peptidylprolyl isomerase A as an endogenous reference gene and were calculated using the ΔΔCt method (Rao et al., 2013).