Total RNA was extracted from frozen tissues or cultured cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. Total RNA was reverse transcribed to cDNA using a PrimeScript™ RT Master Mix (Perfect Real Time) cDNA synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. Amplifications were performed in a quantitative real-time PCR machine (Agilent Technologies, Inc., Santa Clara, CA, USA) using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.). The PCR cycling conditions were conducted as follows: 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec and 60°C for 31 sec. The primer sequences used were as follows: Human β-actin forward, 5′-AGCGAGCATCCCCCAAAGTT-3′; and reverse, 5′-GGGCACGAAGGCTCATCATT-3′ (15 (link)); human PYCR1 forward, 5′-ACACCCCACAACAAGGAGAC-3′; and reverse, 5′-CTGGAGTGTTGGTCATGCAG-3′ (16 (link)). All samples were loaded in triplicates. Relative mRNA expression levels were compared via the 2−ΔΔCq method or log-transformed (17 (link)). The data were analyzed using MxPro qPCR software v1.2 (Agilent Technologies, Inc.).